52906, 33408, and 12189, novel potassium channel family members and uses thereof

ABSTRACT

The invention provides isolated nucleic acids molecules, designated 52906, 33408, or 12189 nucleic acid molecules, which encode novel potassium channel members. The invention also provides antisense nucleic acid molecules, recombinant expression vectors containing 52906, 33408, or 12189 nucleic acid molecules, host cells into which the expression vectors have been introduced, and nonhuman transgenic animals in which a 52906, 33408, or 12189 gene has been introduced or disrupted. The invention still further provides isolated 52906, 33408, or 12189 proteins, fusion proteins, antigenic peptides and anti-52906, 33408, or 12189 antibodies. Diagnostic methods utilizing compositions of the invention are also provided.

RELATED APPLICATIONS

This application claims priority to U.S. provisional application No.60/209,845 filed on Jun. 6, 2000, the contents of which are incorporatedherein by reference.

BACKGROUND OF THE INVENTION

Potassium (K⁺) channels are ubiquitous proteins which are involved inthe setting of the resting membrane potential as well as in themodulation of the electrical activity of cells. In excitable cells, K⁺channels influence action potential waveforms, firing frequency, andneurotransmitter secretion (Rudy, B. (1988) Neuroscience, 25, 729-749;Hille, B. (1992) Ionic Channels of Excitable Membranes, 2nd Ed.). Innon-excitable cells, they are involved in hormone secretion, cell volumeregulation and potentially in cell proliferation and differentiation(Lewis et al. (1995) Annu. Rev. Immunol., 13, 623-653). Developments inelectrophysiology have allowed the identification and thecharacterization of an astonishing variety of K⁺ channels that differ intheir biophysical properties, pharmacology, regulation and tissuedistribution (Rudy, B. (1988) Neuroscience, 25, 729-749; Hille, B.(1992) Ionic Channels of Excitable Membranes, 2nd Ed.). More recently,cloning efforts have shed considerable light on the mechanisms thatdetermine this functional diversity. Furthermore, analyses ofstructure-function relationships have provided an important set of dataconcerning the molecular basis of the biophysical properties(selectivity, gating, assembly) and the pharmacological properties ofcloned K⁺ channels.

Functional diversity of K⁺ channels arises mainly from the existence ofa great number of genes coding for pore-forming subunits, as well as forother associated regulatory subunits. Two main structural families ofpore-forming subunits have been identified. The first one consists ofsubunits with a conserved hydrophobic core containing six transmembranedomains (TMDs). These K⁺ channel α subunits participate in the formationof outward rectifier voltage-gated (Kv) and Ca²⁺-dependent K⁺ channels.The fourth TMD contains repeated positive charges involved in thevoltage gating of these channels and hence in their outwardrectification (Logothetis et al. (1992) Neuron, 8,531-540; Bezanilla etal. (1994) Biophys. J. 66, 1011-1021).

The second family of pore-forming subunits have only two TMDs. They areessential subunits of inward-rectifying (IRK), G-protein-coupled (GIRK)and ATP-sensitive (K_(ATP)) K⁺ channels. The inward rectificationresults from a voltage-dependent block by cytoplasmic Mg²⁺ andpolyamines (Matsuda, H. (1991) Annu. Rev. Physiol., 53, 289-298). Aconserved domain, called the P domain, is present in all members of bothfamilies (Pongs, O. (1993) J. Membr. Biol., 136, 1-8; Heginbotham et al.(1994) Biophys. J. 66,1061-1067; Mackinnon, R. (1995) Neuron, 14,889-892; Pascual et al., (1995) Neuron., and 14, 1055-1063). This domainis an essential element of the aqueous K⁺-selective pore. In bothgroups, the assembly of four subunits is necessary to form a functionalK⁺ channel (Mackinnon, R. (1991) Nature, 350, 232-235; Yang et al.,(1995) Neuron, 15, 1441-1447.

In both six TMD and two TMD pore-forming subunit families, differentsubunits coded by different genes can associate to form heterotetramerswith new channel properties (Isacoff et al., (1990) Nature, 345,530-534). A selective formation of heteropolymeric channels may alloweach cell to develop the best K⁺ current repertoire suited to itsfunction. Pore-forming α subunits of Kv channels are classified intodifferent subfamilies according to their sequence similarity (Chandy etal. (1993) Trends Pharmacol. Sci., 14, 434). Tetramerization is believedto occur preferentially between members of each subgroup (Covarrubias etal. (1991) Neuron, 7, 763-773). The domain responsible for thisselective association is localized in the N-terminal region and isconserved between members of the same subgroup. This domain is necessaryfor hetero-but not homomultimeric assembly within a subfamily andprevents co-assembly between subfamilies. Recently, pore-formingsubunits with two TMDs were also shown to co-assemble to formheteropolymers (Duprat et al. (1995) Biochem. Biophys. Res. Commun.,212, 657-663. This heteropolymerization seems necessary to givefunctional GIRKs. IRKs are active as homopolymers but also formheteropolymers.

SUMMARY OF THE INVENTION

The present invention is based, in part, on the discovery of novelpotassium channel family members, referred to herein as “52906,”“33408,” and “12189.” The nucleotide sequence of a cDNA encoding 52906is shown in SEQ ID NO:1, and the amino acid sequence of a 52906polypeptide is shown in SEQ ID NO:2. In addition, the nucleotidesequences of the coding region are depicted in SEQ ID NO:3. Thenucleotide sequence of a cDNA encoding 33408 is shown in SEQ ID NO:4,and the amino acid sequence of a 33408 polypeptide is shown in SEQ IDNO:5. In addition, the nucleotide sequences of the coding region aredepicted in SEQ ID NO:6. The nucleotide sequence of a cDNA encoding12189 is shown in SEQ ID NO:7, and the amino acid sequence of a 12189polypeptide is shown in SEQ ID NO:8. In addition, the nucleotidesequences of the coding region are depicted in SEQ ID NO:7.

Accordingly, in one aspect, the invention features a nucleic acidmolecule that encodes a 52906, 33408, or 12189 protein or polypeptide,e.g., a biologically active portion of the 52906, 33408, or 12189protein. In a preferred embodiment the isolated nucleic acid moleculeencodes a polypeptide having the amino acid sequence of SEQ ID NO:2, SEQID NO:5, or SEQ ID NO:8. In other embodiments, the invention providesisolated 52906, 33408, or 12189 nucleic acid molecules having thenucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQID NO:6, SEQ ID NO:7, the sequence of the DNA insert of the plasmiddeposited with ATCC Accession Number ______, the sequence of the DNAinsert of the plasmid deposited with ATCC Accession Number ______, orthe sequence of the DNA insert of the plasmid deposited with ATCCAccession Number ______. In still other embodiments, the inventionprovides nucleic acid molecules that are substantially identical (e.g.,naturally occurring allelic variants) to the nucleotide sequence shownin SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, thesequence of the DNA insert of the plasmid deposited with ATCC AccessionNumber ______, the sequence of the DNA insert of the plasmid depositedwith ATCC Accession Number ______, or the sequence of the DNA insert ofthe plasmid deposited with ATCC Accession Number ______. In otherembodiments, the invention provides a nucleic acid molecule whichhybridizes under a stringency condition described herein to a nucleicacid molecule comprising the nucleotide sequence of SEQ ID NO:1, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, the sequence of the DNAinsert of the plasmid deposited with ATCC Accession Number ______, thesequence of the DNA insert of the plasmid deposited with ATCC AccessionNumber ______, or the sequence of the DNA insert of the plasmiddeposited with ATCC Accession Number ______, wherein the nucleic acidencodes a full length 52906, 33408, or 12189 protein or an activefragment thereof.

In a related aspect, the invention further provides nucleic acidconstructs that include a 52906, 33408, or 12189 nucleic acid moleculedescribed herein. In certain embodiments, the nucleic acid molecules ofthe invention are operatively linked to native or heterologousregulatory sequences. Also included, are vectors and host cellscontaining the 52906, 33408, or 12189 nucleic acid molecules of theinvention e.g., vectors and host cells suitable for producing 52906,33408, or 12189 nucleic acid molecules and polypeptides.

In another related aspect, the invention provides nucleic acid fragmentssuitable as primers or hybridization probes for the detection of 52906,33408, or 12189-encoding nucleic acids.

In still another related aspect, isolated nucleic acid molecules thatare antisense to a 52906, 33408, or 12189 encoding nucleic acid moleculeare provided.

In another aspect, the invention features, 52906, 33408, or 12189polypeptides, and biologically active or antigenic fragments thereofthat are useful, e.g., as reagents or targets in assays applicable totreatment and diagnosis of 52906, 33408, or 12189-mediated or -relateddisorders. In another embodiment, the invention provides 52906, 33408,or 12189 polypeptides having a 52906, 33408, or 12189 activity.Preferred polypeptides are 52906, 33408, or 12189 proteins including atleast one ion transport protein domain, and, preferably, having a 52906,33408, or 12189 activity, e.g., a 52906, 33408, or 12189 activity asdescribed herein.

In other embodiments, the invention provides 52906, 33408, or 12189polypeptides, e.g., a 52906, 33408, or 12189 polypeptide having theamino acid sequence shown in SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, theamino acid sequence encoded by the cDNA insert of the plasmid depositedwith ATCC Accession Number ______, the amino acid sequence encoded bythe cDNA insert of the plasmid deposited with ATCC Accession Number______, or the amino acid sequence encoded by the cDNA insert of theplasmid deposited with ATCC Accession Number ______; an amino acidsequence that is substantially identical to the amino acid sequenceshown in SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, the amino acid sequenceencoded by the cDNA insert of the plasmid deposited with ATCC AccessionNumber ______, the amino acid sequence encoded by the cDNA insert of theplasmid deposited with ATCC Accession Number ______, or the amino acidsequence encoded by the cDNA insert of the plasmid deposited with ATCCAccession Number ______; or an amino acid sequence encoded by a nucleicacid molecule having a nucleotide sequence which hybridizes under astringency condition described herein to a nucleic acid moleculecomprising the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:6, SEQ ID NO:7, the sequence of the DNA insert of theplasmid deposited with ATCC Accession Number ______, the sequence of theDNA insert of the plasmid deposited with ATCC Accession Number ______,or the sequence of the DNA insert of the plasmid deposited with ATCCAccession Number ______, wherein the nucleic acid encodes a full length52906, 33408, or 12189 protein or an active fragment thereof.

In a related aspect, the invention further provides nucleic acidconstructs which include a 52906, 33408, or 12189 nucleic acid moleculedescribed herein.

In a related aspect, the invention provides 52906, 33408, or 12189polypeptides or fragments operatively linked to non-52906, 33408, or12189 polypeptides to form fusion proteins.

In another aspect, the invention features antibodies and antigen-bindingfragments thereof, that react with, or more preferably specifically bind52906, 33408, or 12189 polypeptides or fragments thereof, e.g., an iontransport protein domain, a cyclic nucleotide-binding domain, apotassium channel tetramerisation domain, a transmembrane domain, acytoplasmic domain, an extracellular domain, a Pore-loop domain, or aPAS domain. In one embodiment, the antibodies or antigen-bindingfragment thereof competitively inhibit the binding of a second antibodyto a 52906, 33408, or 12189 polypeptide or a fragment thereof, e.g., anion transport protein domain, a cyclic nucleotide-binding domain, apotassium channel tetramerisation domain, a transmembrane domain, acytoplasmic domain, an extracellular domain, a Pore-loop domain, or aPAS domain.

In another aspect, the invention provides methods of screening forcompounds that modulate the expression or activity of the 52906, 33408,or 12189 polypeptides or nucleic acids.

In still another aspect, the invention provides a process for modulating52906, 33408, or 12189 polypeptide or nucleic acid expression oractivity, e.g. using the screened compounds. In certain embodiments, themethods involve treatment of conditions related to aberrant activity orexpression of the 52906, 33408, or 12189 polypeptides or nucleic acids,such as conditions characterized by abnormal ion flux such as aneurological disorder or a cardiac disorder.

The invention also provides assays for determining the activity of orthe presence or absence of 52906, 33408, or 12189 polypeptides ornucleic acid molecules in a biological sample, including for diseasediagnosis.

In yet another aspect, the invention provides methods for modulating(increasing or decreasing) the ion flux, e.g., the flow of K⁺ ions, in a52906, 33408, or 12189-expressing cell. The method includes contactingthe cell with a compound (e.g., a compound identified using the methodsdescribed herein) that modulates the activity, or expression, of the52906, 33408, or 12189 polypeptide or nucleic acid. In a preferredembodiment, the contacting step is effective in vitro or ex vivo. Inother embodiments, the contacting step is effected in vivo, e.g., in asubject (e.g., a mammal, e.g., a human), as part of a therapeutic orprophylactic protocol. In a preferred embodiment, the cell is anelectrically excitable cell, e.g., a neuronal cell or a muscle cell(e.g., a heart cell). For example, the cell can be from brain or cardiactissues.

In a preferred embodiment, the compound is an inhibitor of a 52906,33408, or 12189 polypeptide. Preferably, the inhibitor is chosen from apeptide, a phosphopeptide, a small organic molecule, a small inorganicmolecule and an antibody (e.g., an antibody conjugated to a therapeuticmoiety). In another preferred embodiment, the compound is an inhibitorof a 52906, 33408, or 12189 nucleic acid, e.g., an antisense, aribozyme, or a triple helix molecule.

In another aspect, the invention features methods for treating orpreventing a disorder characterized by the abnormal ion flux of a 52906,33408, or 12189-expressing cell, in a subject. Preferably, the methodincludes administering to the subject (e.g., a mammal, e.g., a human) aneffective amount of a compound (e.g., a compound identified using themethods described herein) that modulates the activity, or expression, ofthe 52906, 33408, or 12189 polypeptide or nucleic acid. In a preferredembodiment, the disorder is a neurological disorder or a cardiacdisorder.

In a further aspect, the invention provides methods for evaluating theefficacy of a treatment of a disorder, e.g., a disorder characterized byabnormal ion flux such as a neurological disorder or a cardiac disorder.The method includes: treating a subject, e.g., a patient or an animal,with a protocol under evaluation (e.g., treating a subject with acompound identified using the methods described herein); and evaluatingthe expression of a 52906, 33408, or 12189 nucleic acid or polypeptidebefore and after treatment. A change, e.g., a decrease or increase, inthe level of a 52906, 33408, or 12189 nucleic acid (e.g., mRNA) orpolypeptide after treatment, relative to the level of expression beforetreatment, is indicative of the efficacy of the treatment of thedisorder. The level of 52906, 33408, or 12189 nucleic acid orpolypeptide expression can be detected by any method described herein.

In a preferred embodiment, the evaluating step includes obtaining asample (e.g., a tissue sample, e.g., a biopsy, or a fluid sample) fromthe subject, before and after treatment and comparing the level ofexpressing of a 52906, 33408, or 12189 nucleic acid (e.g., mRNA) orpolypeptide before and after treatment.

In another aspect, the invention provides methods for evaluating theefficacy of a therapeutic or prophylactic agent. The method includes:contacting a sample with an agent (e.g., a compound identified using themethods described herein) and, evaluating the expression of 52906,33408, or 12189 nucleic acid or polypeptide in the sample before andafter the contacting step. A change, e.g., a decrease or increase, inthe level of 52906, 33408, or 12189 nucleic acid (e.g., mRNA) orpolypeptide in the sample obtained after the contacting step, relativeto the level of expression in the sample before the contacting step, isindicative of the efficacy of the agent. The level of 52906, 33408, or12189 nucleic acid or polypeptide expression can be detected by anymethod described herein. In a preferred embodiment, the sample includesneuronal cells or muscle cells.

In further aspect, the invention provides assays for determining thepresence or absence of a genetic alteration in a 52906, 33408, or 12189polypeptide or nucleic acid molecule, including for disease diagnosis.

In another aspect, the invention features a two dimensional array havinga plurality of addresses, each address of the plurality beingpositionally distinguishable from each other address of the plurality,and each address of the plurality having a unique capture probe, e.g., anucleic acid or peptide sequence. At least one address of the pluralityhas a capture probe that recognizes a 52906, 33408, or 12189 molecule.In one embodiment, the capture probe is a nucleic acid, e.g., a probecomplementary to a 52906, 33408, or 12189 nucleic acid sequence. Inanother embodiment, the capture probe is a polypeptide, e.g., anantibody specific for 52906, 33408, or 12189 polypeptides. Also featuredis a method of analyzing a sample by contacting the sample to theaforementioned array and detecting binding of the sample to the array.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a hydropathy plot of human 52906. Relative hydrophobicresidues are shown above the dashed horizontal line, and relativehydrophilic residues are below the dashed horizontal line. Numberscorresponding to positions in the amino acid sequence of human 52906 areindicated. Polypeptides of the invention include fragments whichinclude: all or part of a hydrophobic sequence, i.e., a sequence abovethe dashed line, e.g., the sequence from about amino acid 785-800 of SEQID NO:2; all or part of a hydrophilic sequence, i.e., a sequence belowthe dashed line, e.g., the sequence of from about amino acid 241-265 ofSEQ ID NO:2.

FIG. 2 depicts an alignment of the ion transport protein domain of human52906 with a consensus amino acid sequence derived from a hidden Markovmodel (HMM) from PFAM. The upper sequence is the consensus amino acidsequence (SEQ ID NO:9), while the lower amino acid sequence correspondsto amino acids 472 to 661 of SEQ ID NO:2.

FIG. 3 depicts a hydropathy plot of human 33408. Relative hydrophobicresidues are shown above the dashed horizontal line, and relativehydrophilic residues are below the dashed horizontal line. Numberscorresponding to positions in the amino acid sequence of human 33408 areindicated. Polypeptides of the invention include fragments whichinclude: all or part of a hydrophobic sequence, i.e., a sequence abovethe dashed line, e.g., the sequence from about amino acid 585-600 of SEQID NO:5; all or part of a hydrophilic sequence, i.e., a sequence belowthe dashed line, e.g., the sequence of from about amino acid 710-740 ofSEQ ID NO:5.

FIG. 4A depicts an alignment of the ion transport protein domain ofhuman 33408 with a consensus amino acid sequence derived from a hiddenMarkov model (HMM) from PFAM. The upper sequence is the consensus aminoacid sequence (SEQ ID NO:9), while the lower amino acid sequencecorresponds to amino acids 247 to 467 of SEQ ID NO:5.

FIG. 4B depicts an alignment of the cyclic nucleotide-binding domain ofhuman 33408 with a consensus amino acid sequence derived from a hiddenMarkov model (HMM) from PFAM. The upper sequence is the consensus aminoacid sequence (SEQ ID NO:10), while the lower amino acid sequencecorresponds to amino acids 565 to 655 of SEQ ID NO:5.

FIGS. 4C-4D depict an alignment of the amino acid sequence of human33408 (upper sequence) with the amino acid sequence of rat Eag2 (lowersequence; Accession Number AF185637; SEQ ID NO:12).

FIG. 5 depicts a hydropathy plot of human 12189. Relative hydrophobicresidues are shown above the dashed horizontal line, and relativehydrophilic residues are below the dashed horizontal line. Numberscorresponding to positions in the amino acid sequence of human 12189 areindicated. Polypeptides of the invention include fragments whichinclude: all or part of a hydrophobic sequence, i.e., a sequence abovethe dashed line, e.g., the sequence from about amino acid 75-95 of SEQID NO:8; all or part of a hydrophilic sequence, i.e., a sequence belowthe dashed line, e.g., the sequence of from about amino acid 35-55 ofSEQ ID NO:8.

FIG. 6A depicts an alignment of the potassium channel tetramerisationdomain of human 12189 with a consensus amino acid sequence derived froma hidden Markov model (HMM) from PFAM. The upper sequence is theconsensus amino acid sequence (SEQ ID NO:11), while the lower amino acidsequence corresponds to amino acids 3 to 101 of SEQ ID NO:8.

FIG. 6B depicts an alignment of the ion transport protein domain ofhuman 12189 with a consensus amino acid sequence derived from a hiddenMarkov model (HMM) from PFAM. The upper sequence is the consensus aminoacid sequence (SEQ ID NO:9), while the lower amino acid sequencecorresponds to amino acids 198 to 383 of SEQ ID NO:8.

FIG. 6C depicts an alignment of the amino acid sequence of human 12189(lower sequence) with the amino acid sequence of mouse Kv1.7 (uppersequence; Accession Number AF032099; SEQ ID NO:13).

DETAILED DESCRIPTION

Human 52906

The human 52906 sequence (see SEQ ID NO:1, as recited in Example 1),which is approximately 3525 nucleotides long including untranslatedregions, contains a predicted methionine-initiated coding sequence ofabout 2544 nucleotides, including the termination codon. The codingsequence encodes a 847 amino acid protein (see SEQ ID NO:2, as recitedin Example 1). The hydropathy plot of 52906 is depicted in FIG. 1.

Human 52906 contains the following regions or structural features: anion transport protein domain (PFAM Accession Number PF00520) located atabout amino acid residues 472 to 661 of SEQ ID NO:2 (see FIG. 2); and acore membrane region consisting of six transmembrane domains, fourcytoplasmic domains, three extracellular domains, and a Pore-loopdomain. The core membrane region is located at about amino acid 402 toabout amino acid 662 of SEQ ID NO:2. The six transmembrane domains arelocated at about amino acid 402 (cytoplasmic end) to about amino acid419 (extracellular end) of SEQ ID NO:2, about amino acid 433(extracellular end) to about amino acid 456 (cytoplasmic end) of SEQ IDNO:2, about amino acid 482 (cytoplasmic end) to about amino acid 498(extracellular end) of SEQ ID NO:2, about amino acid 524 (extracellularend) to about amino acid 543 (cytoplasmic end) of SEQ ID NO:2, aboutamino acid 573 (cytoplasmic end) to about amino acid 597 (extracellularend) of SEQ ID NO:2, and about amino acid 641 (extracellular end) toabout amino acid 662 (cytoplasmic end) of SEQ ID NO:2. The fourcytoplasmic domains are located at about amino acids 1 to 401 (aminoterminus), 457 to 481, 544 to 572, and 663 to 847 (carboxy terminus) ofSEQ ID NO:2. The three extracellular domains are located at about aminoacids 420 to 432, 499 to 523, and 598 to 640 of SEQ ID NO:2. Theextracellular domain located at about amino acids 598 to 640 includes aPore-loop domain (P-loop domain) located at about amino acid residues616 to 639 of SEQ ID NO:2.

The 52906 protein also includes the following domains: six predictedN-glycosylation sites (PS00001) located at about amino acids 10-13,141-144, 182-185, 284-287, 342-345, and 500-503 of SEQ ID NO:2; onepredicted glycosaminoglycan attachment site (PS00002) located at aboutamino acids 367-370 of SEQ ID NO:2; four predicted cAMP- andcGMP-dependent protein kinase phosphorylation sites (PS00004) located atabout amino acids 176-179, 258-261, 400-403, and 832-835 of SEQ ID NO:2;13 predicted Protein Kinase C phosphorylation sites (PS00005) located atabout amino acids 9-11, 12-14, 174-176, 271-273, 288-290, 377-379,506-508, 552-554, 596-598, 684-686, 732-734, 799-801, and 829-831 of SEQID NO:2; seven predicted Casein Kinase II phosphorylation sites(PS00006) located at about amino acids 330-333, 337-340, 518-521,668-671, 746-749, 780-783, and 842-845 of SEQ ID NO:2; 15 predictedN-myristoylation sites (PS00008) located at about amino acids 21-26,42-47, 118-123, 132-137, 153-158, 165-170, 178-183, 227-232, 309-314,351-356, 359-364, 366-371, 374-379, 647-652, and 787-792 of SEQ ID NO:2;and one predicted coiled coil located at about amino acids 719-791 ofSEQ ID NO:2.

For general information regarding PFAM identifiers, PS prefix and PFprefix domain identification numbers, refer to Sonnhammer et al. (1997)Protein 28:405-420 andhttp://www.psc.edu/general/software/packages/pfam/pfam.html.

A plasmid containing the nucleotide sequence encoding human 52906 (clone“Fbh52906FL”) was deposited with American Type Culture Collection(ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209, on ______and assigned Accession Number ______. This deposit will be maintainedunder the terms of the Budapest Treaty on the International Recognitionof the Deposit of Microorganisms for the Purposes of Patent Procedure.This deposit was made merely as a convenience for those of skill in theart and is not an admission that a deposit is required under 35 U.S.C.§112.

An alignment of the human 52906 amino acid sequence with the rat SK2amino acid sequence (Accession Number U69882) suggests that 52906 is ahuman ortholog of rat SK2, a calcium activated potassium channel (Köhleret al. (1996) Science 273:1709-1714).

Human 33408

The human 33408 sequence (see SEQ ID NO:4, as recited in Example 1),which is approximately 3553 nucleotides long including untranslatedregions, contains a predicted methionine-initiated coding sequence ofabout 2967 nucleotides, including the termination codon. The codingsequence encodes a 988 amino acid protein (see SEQ ID NO:5, as recitedin Example 1). The hydropathy plot of 33408 is depicted in FIG. 3.

Human 33408 contains the following regions or structural features: anion transport protein domain (PFAM Accession Number. PF00520) located atabout amino acid residues 247 to 467 of SEQ ID NO:5 (see FIG. 4A); acyclic nucleotide-binding domain (PFAM Accession Number PF00027) locatedat about amino acid residues 565 to 655 of SEQ ID NO:5 (see FIG. 4B);and a core membrane region consisting of six transmembrane domains, fourcytoplasmic domains, three extracellular domains, a Pore-loop domain,and a PAS domain. The core membrane region is located at about aminoacid 219 to about amino acid 471 of SEQ ID NO:5. The six transmembranedomains are located at about amino acid 219 (cytoplasmic end) to aboutamino acid 236 (extracellular end) of SEQ ID NO:5, about amino acid 245(extracellular end) to about amino acid 264 (cytoplasmic end) of SEQ IDNO:5, about amino acid 292 (cytoplasmic end) to about amino acid 309(extracellular end) of SEQ ID NO:5, about amino acid 320 (extracellularend) to about amino acid 337 (cytoplasmic end) of SEQ ID NO:5, aboutamino acid 344 (cytoplasmic end) to about amino acid 368 (extracellularend) of SEQ ID NO:5, and about amino acid 447 (extracellular end) toabout amino acid 471 (cytoplasmic end) of SEQ ID NO:5. The fourcytoplasmic domains are located at about amino acids 1 to 218 (aminoterminus), 265 to 291, 338 to 343, and 472 to 988 (carboxy terminus) ofSEQ ID NO:5. The three extracellular domains are located at about aminoacids 237 to 244, 310 to 319, and 369 to 446 of SEQ ID NO:5. Theextracellular domain located at about amino acids 369 to 446 includes aPore-loop domain (P-loop domain) located at about amino acid residues420 to 440 of SEQ ID NO:5. The cytoplasmic domain located at about aminoacids 1 to 218 includes a PAS domain located at about amino acidresidues 1-134 of SEQ ID NO:5 and a PAC domain located at about aminoacid residues 92-132 of SEQ ID NO:5.

The 33408 protein also includes the following domains: seven predictedN-glycosylation sites (PS00001) located at about amino acids 170-173,235-238, 403-406, 466-469, 663-666, 743-746, and 830-833 of SEQ ID NO:5;two predicted cAMP- and cGMP-dependent protein kinase phosphorylationsites (PS00004) located at about amino acids 21-24 and 677-680 of SEQ IDNO:5; 13 predicted Protein Kinase C phosphorylation sites (PS00005)located at about amino acids 73-75, 127-129, 142-144, 237-239, 322-324,478-480, 502-504, 521-523, 773-775, 925-927, 943-945, 952-954, and981-983 of SEQ ID NO:5; 16 predicted Casein Kinase II phosphorylationsites (PS00006) located at about amino acids 14-17, 127-130, 215-218,252-255, 369-372, 442-445, 634-637, 725-728, 832-835, 847-850, 869-872,883-886, 909-912, 929-932, 974-977, and 981-984 of SEQ ID NO:5; eightpredicted N-myristoylation sites (PS00008) located at about amino acids3-8, 407-412, 465-470, 557-562, 723-728, 744-749, 806-811, and 867-872of SEQ ID NO:5; one predicted amidation site (PS00009) located at aboutamino acids 3-6 of SEQ ID NO:5; one predicted leucine zipper pattern(PS00029) located at about amino acids 910-931 of SEQ ID NO:5; and onepredicted coiled coil located at about amino acids 906-944 of SEQ IDNO:5.

A plasmid containing the nucleotide sequence encoding human 33408 (clone“Fbh33408FL”) was deposited with American Type Culture Collection(ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209, on ______and assigned Accession Number ______. This deposit will be maintainedunder the terms of the Budapest Treaty on the International Recognitionof the Deposit of Microorganisms for the Purposes of Patent Procedure.This deposit was made merely as a convenience for those of skill in theart and is not an admission that a deposit is required under 35 U.S.C.§112.

An alignment of the human 33408 amino acid sequence with the rat Eag2amino acid sequence (SEQ ID NO:12; Accession Number AF185637) isdepicted in FIGS. 4C-4D. 33408 appears to be a human ortholog of ratEag2, a subthreshold activating potassium channel (Saganich et al.(1999) J. Neuroscience 19:10789-10802).

Human 12189

The human 12189 sequence (see SEQ ID NO:7, as recited in Example 1),which is approximately 1341 nucleotides long, contains a predictedcoding sequence, including a termination codon. The coding sequenceencodes a 446 amino acid protein (see SEQ ID NO:8, as recited in Example1). The hydropathy plot of 12189 is depicted in FIG. 5.

Human 12189 contains the following regions or structural features: apotassium channel tetramerisation domain (PFAM Accession Number PF02214)located at about amino acid residues 3 to 101 of SEQ ID NO:8 (see FIG.6A); an ion transport protein domain (PFAM Accession Number PF00520)located at about amino acid residues 198 to 383 of SEQ ID NO:8 (see FIG.6B); and a core membrane region consisting of six transmembrane domains,four cytoplasmic domains, three extracellular domains, and a Pore-loopdomain. The core membrane region is located at about amino acid 134 toabout amino acid 384 of SEQ ID NO:8. The six transmembrane domains arelocated at about amino acid 134 (cytoplasmic end) to about amino acid152 (extracellular end) of SEQ ID NO:8, about amino acid 200(extracellular end) to about amino acid 222 (cytoplasmic end) of SEQ IDNO:8, about amino acid 231 (cytoplasmic end) to about amino acid 248(extracellular end) of SEQ ID NO:8, about amino acid 266 (extracellularend) to about amino acid 286 (cytoplasmic end) of SEQ ID NO:8, aboutamino acid 302 (cytoplasmic end) to about amino acid 323 (extracellularend) of SEQ ID NO:8, and about amino acid 363 (extracellular end) toabout amino acid 384 (cytoplasmic end) of SEQ ID NO:8. The fourcytoplasmic domains are located at about amino acids 1 to 133 (aminoterminus), 223 to 230, 287 to 301, and 385 to 446 (carboxy terminus) ofSEQ ID NO:8. The three extracellular domains are located at about aminoacids 153 to 199, 249 to 265, and 324 to 362 of SEQ ID NO:8. Theextracellular domain located at about amino acids 324 to 362 includes aPore-loop domain (P-loop domain) located at about amino acid residues339 to 355 of SEQ ID NO:8.

The 12189 protein also includes the following domains: two predictedN-glycosylation sites (PS00001) located at about amino acids 181-184 and386-389 of SEQ ID NO:8; two predicted Protein Kinase C phosphorylationsites (PS00005) located at about amino acids 294-296 and 298-300 of SEQID NO:8; five predicted Casein Kinase II phosphorylation sites (PS00006)located at about amino acids 154-157, 298-301, 334-337, 395-398, and404-407 of SEQ ID NO:8; one predicted tyrosine kinase phosphorylationsite (PS00007) located at about amino acids 52-60 of SEQ ID NO:8; fivepredicted N-myristoylation sites (PS00008) located at about amino acids87-92, 164-169, 248-253, 365-370, and 421-426 of SEQ ID NO:8; and onepredicted leucine zipper pattern (PS00029) located at about amino acids281-302 of SEQ ID NO:8.

A plasmid containing the nucleotide sequence encoding human 12189 (clone“Fbh12189 FL”) was deposited with American Type Culture Collection(ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209, on ______and assigned Accession Number ______. This deposit will be maintainedunder the terms of the Budapest Treaty on the International Recognitionof the Deposit of Microorganisms for the Purposes of Patent Procedure.This deposit was made merely as a convenience for those of skill in theart and is not an admission that a deposit is required under 35 U.S.C.§112.

An alignment of the human 12189 amino acid sequence with the mouse Kv1.7amino acid sequence (SEQ ID NO:13; Accession Number AF032099) isdepicted in FIG. 6C. 12189 appears to be a human ortholog of mouseKv1.7, a voltage-gated potassium channel (Kalman et al. (1998) J. Biol.Chem. 273:5851-5857). TABLE 1 Summary of Sequence Information for 52906,33408, and 12189 ATCC Accession Gene cDNA ORF Polypeptide Number 52906SEQ ID NO: 1 SEQ ID NO: 3 SEQ ID NO: 2 33408 SEQ ID NO: 4 SEQ ID NO: 6SEQ ID NO: 5 12189 SEQ ID NO: 7 SEQ ID NO: 8

TABLE 2 Summary of Domains of 52906, 33408, and 12189 Domain 52906 3340812189 Transmembrane amino acids 402-662 amino acids 219-471 amino acids134-384 Region of SEQ ID NO: 2 of SEQ ID NO: 5 of SEQ ID NO: 8Transmembrane amino acids 402-419 amino acids 219-236 amino acids134-152 Domain 1 of SEQ ID NO: 2 of SEQ ID NO: 5 of SEQ ID NO: 8Transmembrane amino acids 433-456 amino acids 245-264 amino acids200-222 Domain 2 of SEQ ID NO: 2 of SEQ ID NO: 5 of SEQ ID NO: 8Transmembrane amino acids 482-498 amino acids 292-309 amino acids231-248 Domain 3 of SEQ ID NO: 2 of SEQ ID NO: 5 of SEQ ID NO: 8Transmembrane amino acids 524-543 amino acids 320-337 amino acids266-286 Domain 4 of SEQ ID NO: 2 of SEQ ID NO: 5 of SEQ ID NO: 8Transmembrane amino acids 573-597 amino acids 344-368 amino acids302-323 Domain 5 of SEQ ID NO: 2 of SEQ ID NO: 5 of SEQ ID NO: 8Transmembrane amino acids 641-662 amino acids 447-471 amino acids363-384 Domain 6 of SEQ ID NO: 2 of SEQ ID NO: 5 of SEQ ID NO: 8Cytoplasmic amino acids 1-401 of amino acids 1-218 of amino acids 1-133of Domain 1 SEQ ID NO: 2 SEQ ID NO: 5 SEQ ID NO: 8 Cytoplasmic aminoacids 457-481 amino acids 265-291 amino acids 223-230 Domain 2 of SEQ IDNO: 2 of SEQ ID NO: 5 of SEQ ID NO: 8 Cytoplasmic amino acids 544-572amino acids 338-343 amino acids 287-301 Domain 3 of SEQ ID NO: 2 of SEQID NO: 5 of SEQ ID NO: 8 Cytoplasmic amino acids 663-847 amino acids472-988 amino acids 385-446 Domain 4 of SEQ ID NO: 2 of SEQ ID NO: 5 ofSEQ ID NO: 8 Extracellular amino acids 420-432 amino acids 237-244 aminoacids 153-199 Domain 1 of SEQ ID NO: 2 of SEQ ID NO: 5 of SEQ ID NO: 8Extracellular amino acids 499-523 amino acids 310-319 amino acids249-265 Domain 2 of SEQ ID NO: 2 of SEQ ID NO: 5 of SEQ ID NO: 8Extracellular amino acids 598-640 amino acids 369-446 amino acids324-362 Domain 3 of SEQ ID NO: 2 of SEQ ID NO: 5 of SEQ ID NO: 8Pore-loop amino acids 616-639 amino acids 420-440 amino acids 339-355Domain of SEQ ID NO: 2 of SEQ ID NO: 5 of SEQ ID NO: 8 ion transportamino acids 472-661 amino acids 247-467 amino acids 198-383 proteindomain of SEQ ID NO: 2 of SEQ ID NO: 5 of SEQ ID NO: 8 cyclic aminoacids 565-655 nucleotide of SEQ ID NO: 5 binding domain potassium aminoacids 3-101 of channel SEQ ID NO: 8 tetramerisation domain

The 52906, 33408, and 12189 proteins contain a significant number ofstructural characteristics in common with members of the potassiumchannel family. The term “family” when referring to the protein andnucleic acid molecules of the invention means two or more proteins ornucleic acid molecules having a common structural domain or motif andhaving sufficient amino acid or nucleotide sequence homology as definedherein. Such family members can be naturally or non-naturally occurringand can be from either the same or different species. For example, afamily can contain a first protein of human origin as well as otherdistinct proteins of human origin, or alternatively, can containhomologues of non-human origin, e.g., rat or mouse proteins. Members ofa family can also have common functional characteristics.

As used herein, a “potassium channel” includes a protein or polypeptidewhich is involved in receiving, conducting, and transmitting signals inan electrically excitable cell, e.g., a neuronal cell or a muscle cell.Potassium channels are potassium ion selective, and can determinemembrane excitability (the ability of, for example, a neuron to respondto a stimulus and convert it into an impulse). Potassium channels canalso influence the resting potential of membranes, wave forms andfrequencies of action potentials, and thresholds of excitation.Potassium channels are typically expressed in electrically excitablecells, e.g., neurons, muscle, endocrine, and egg cells, and may formheteromultimeric structures, e.g., composed of pore-forming α andcytoplasmic β subunits. Potassium channels may also be found innonexcitable cells (e.g., thymus cells), where they may play a role in,e.g., signal transduction. Potassium channel proteins contain sixtransmembrane helices, wherein the last two helices flank a loop (aP-loop) which determines potassium ion selectivity. Examples ofpotassium channels include: (1) the voltage-gated potassium channels,(2) the ligand-gated potassium channels, e.g., neurotransmitter-gatedpotassium channels, and (3) cyclic-nucleotide-gated potassium channels.Voltage-gated and ligand-gated potassium channels are expressed in thebrain, e.g., in brainstem monoaminergic and forebrain cholinergicneurons, where they are involved in the release of neurotransmitters, orin the dendrites of hippocampal and neocortical pyramidal cells, wherethey are involved in the processes of learning and memory formation. Fora detailed description of potassium channels, see Kandel E. R. et al.,Principles of Neural Science, second edition, (Elsevier SciencePublishing Co., Inc., N.Y. (1985)), the contents of which areincorporated herein by reference.

A 52906, 33408, or 12189 polypeptide can include a “transmembranedomain” or regions homologous with a “transmembrane domain”.

As used herein, the term “transmembrane domain” includes an amino acidsequence of about 15 amino acid residues in length which spans theplasma membrane. More preferably, a transmembrane domain includes aboutat least 20, 25, 30, 35, 40, or 45 amino acid residues and spans theplasma membrane. Transmembrane domains are rich in hydrophobic residues,and typically have an alpha-helical structure. In a preferredembodiment, at least 50%, 60%, 70%, 80%, 90%, 95% or more of the aminoacids of a transmembrane domain are hydrophobic, e.g., leucines,isoleucines, tyrosines, or tryptophans. Transmembrane domains aredescribed in, for example, Zagotta W. N. et al., (1996) Annual Rev.Neurosci. 19: 235-263, the contents of which are incorporated herein byreference. Amino acid residues 402-419, 433-456, 482-498, 524-543,573-597, and 641-662 of the 52906 protein (SEQ ID NO:2) are predicted tocomprise transmembrane domains (see FIG. 2). Accordingly, 52906 proteinshaving at least 50-60% homology, preferably about 60-70%, morepreferably about 70-80%, or about 80-90% homology with a transmembranedomain of human 52906 are within the scope of the invention. Amino acidresidues 219-236, 245-264, 292-309, 320-337, 344-368, and 447-471 of the33408 protein (SEQ ID NO:5) are predicted to comprise transmembranedomains (see FIG. 5). Accordingly, 33408 proteins having at least 50-60%homology, preferably about 60-70%, more preferably about 70-80%, orabout 80-90% homology with a transmembrane domain of human 33408 arewithin the scope of the invention. Amino acid residues 134-152, 200-222,231-248, 266-286, 302-323, and 363-384 of the 12189 protein (SEQ IDNO:8) are predicted to comprise transmembrane domains (see FIG. 8).Accordingly, 12189 proteins having at least 50-60% homology, preferablyabout 60-70%, more preferably about 70-80%, or about 80-90% homologywith a transmembrane domain of human 12189 are within the scope of theinvention.

A 52906, 33408, or 12189 polypeptide can further include a “Pore loop”or regions homologous with a “Pore loop domain”.

As used herein, the term “Pore loop” or “P-loop” includes amino acidsequence of about 15-45 amino acid residues in length, preferably about15-35 amino acid residues in length, and most preferably about 15-25amino acid residues in length, which is hydrophobic and which isinvolved in lining the potassium channel pore. A P-loop is typicallyfound between transmembrane domains of potassium channels and isbelieved to be a major determinant of ion selectivity in potassiumchannels. Preferably, P-loops contain a G-[HYDROPHOBIC AMINO ACID]-Gsequence, e.g., a GYG, GLG, or GFG sequence. P-loops are described in,for example, Warmke et al. (1991) Science 252:1560-1562; Zagotta W. N.et al., (1996) Annual Rev. Neuronsci. 19:235-63 (Pongs, O. (1993) J.Membr. Biol., 136, 1-8; Heginbotham et al. (1994) Biophys. J.66,1061-1067; Mackinnon, R. (1995) Neuron, and 14, 889-892; Pascual etal., (1995) Neuron., 14, 1055-1063), the contents of which areincorporated herein by reference. Amino acid residues 616-639 of SEQ IDNO:2, 420-440 of SEQ ID NO:5, and 339-355 of SEQ ID NO:8 comprise P-loopdomains. Accordingly, proteins having at least 50-60% homology,preferably about 60-70%, more preferably about 70-80%, or about 80-90%homology with a -loop domain of human 52906, 33408, or 12189 are withinthe scope of the invention.

In one embodiment, a 52906, 33408, or 12189 protein includes at leastone cytoplasmic domain. When located at the N-terminal domain thecytoplasmic domain is referred to herein as an “N-terminal cytoplasmicdomain”. As used herein, an “N-terminal cytoplasmic domain” includes anamino acid sequence having about 1-500, preferably about 1-450,preferably about 1-400, preferably about 1-380, more preferably about1-350, more preferably about 1-300, more preferably about 1-220, or evenmore preferably about 1-135 amino acid residues in length and is locatedinside of a cell or intracellularly. The C-terminal amino acid residueof a “N-terminal cytoplasmic domain” is adjacent to an N-terminal aminoacid residue of a transmembrane domain in a 52906, 33408, or 12189protein. For example, an N-terminal cytoplasmic domain is located atabout amino acid residues 1-401 of SEQ ID NO:2, 1-218 of SEQ ID NO:5, or1-133 of SEQ ID NO:8.

In a preferred embodiment, a 52906, 33408, or 12189 polypeptide orprotein has at least one cytoplasmic domain or a region which includesat least about 5, preferably about 5-10, and more preferably about 10-20amino acid residues and has at least about 60%, 70% 80% 90% 95%, 99%, or100% homology with an “cytoplasmic domain,” e.g., at least onecytoplasmic domain of human 52906, 33408, or 12189 (e.g., residues1-401, 457-481, 544-572, and 663-847 of SEQ ID NO:2; residues 1-218,265-291, 338-343, and 447-988 of SEQ ID NO:5; and residues 1-133,223-230, 287-301, and 385-446 of SEQ ID NO:8).

In another embodiment, a 52906, 33408, or 12189 protein includes atleast one extracellular loop. As used herein, the term “loop” includesan amino acid sequence having a length of at least about 4, preferablyabout 5-10, and more preferably about 10-20 amino acid residues, and hasan amino acid sequence that connects two transmembrane domains within aprotein or polypeptide. Accordingly, the N-terminal amino acid of a loopis adjacent to a C-terminal amino acid of a transmembrane domain in a52906, 33408, or 12189 molecule, and the C-terminal amino acid of a loopis adjacent to an N-terminal amino acid of a transmembrane domain in a52906, 33408, or 12189 molecule. As used herein, an “extracellular loop”includes an amino acid sequence located outside of a cell, orextracellularly. For example, an extracellular loop can be found atabout amino acids 420-432, 499-523, and 598-640 of SEQ ID NO:2; at aboutamino acids 237-244, 310-319, and 369-446 of SEQ ID NO:5; and at aboutamino acids 153-199, 249-265, and 324-362 of SEQ ID NO:8.

In a preferred embodiment, a 52906, 33408, or 12189 polypeptide orprotein has at least one extracellular loop or a region which includesat least about 4, preferably about 5-10, preferably about 10-20, andmore preferably about 20-30 amino acid residues and has at least about60%, 70% 80% 90% 95%, 99%, or 100% homology with an “extracellularloop,” e.g., at least one extracellular loop of human 52906, 33408, or12189 (e.g., residues 420-432, 499-523, and 598-640 of SEQ ID NO:2;residues 237-244, 310-319, and 369-446 of SEQ ID NO:5; and residues153-199, 249-265, and 324-362 of SEQ ID NO:8).

In another embodiment, a 52906, 33408, or 12189 protein includes a“C-terminal cytoplasmic domain”, also referred to herein as a C-terminalcytoplasmic tail, in the sequence of the protein. As used herein, a“C-terminal cytoplasmic domain” includes an amino acid sequence having alength of at least about 50, preferably about 500-550, preferably about150-200, more preferably about 50-70 amino acid residues and is locatedwithin a cell or within the cytoplasm of a cell. Accordingly, theN-terminal amino acid residue of a “C-terminal cytoplasmic domain” isadjacent to a C-terminal amino acid residue of a transmembrane domain ina 52906, 33408, or 12189 protein. For example, a C-terminal cytoplasmicdomain is found at about amino acid residues 663-847 of SEQ ID NO:2; atabout amino acid residues 472-988 of SEQ ID NO:5; and at about aminoacid residues 385-446 of SEQ ID NO:8.

In a preferred embodiment, a 52906, 33408, or 12189 polypeptide orprotein has a C-terminal cytoplasmic domain or a region which includesat least about 50, preferably about 150-550, more preferably about 50-70amino acid residues and has at least about 60%, 70% 80% 90% 95%, 99%, or100% homology with an “C-terminal cytoplasmic domain,” e.g., theC-terminal cytoplasmic domain of human 52906, 33408, or 12189 (e.g.,residues 663-847 of SEQ ID NO:2; residues 472-988 of SEQ ID NO:5; andresidues 385-446 of SEQ ID NO:8).

A 52906, 33408, or 12189 polypeptide can include an “ion transportprotein domain” or regions homologous with an “ion transport proteindomain.”

As used herein, the term “ion transport protein domain” includes anamino acid sequence of about 100 to 300 amino acid residues in lengthand having a bit score for the alignment of the sequence to the iontransport protein domain profile (Pfam HMM) of at least 50. Preferably,a ion transport protein domain includes at least about 150 to 280 aminoacids, more preferably about 170 to 260 amino acid residues, or about180 to 230 amino acids and has a bit score for the alignment of thesequence to the ion transport protein domain (HMM) of at least 90 orgreater. The ion transport protein domain (HMM) has been assigned thePFAM Accession Number PF00520 (http;//genome.wustl.edu/Pfam/.html). Analignment of the ion transport protein domain (amino acids 472-661 ofSEQ ID NO:2) of human 52906 with a consensus amino acid sequence (SEQ IDNO:9) derived from a hidden Markov model is depicted in FIG. 2. Analignment of the ion transport protein domain (amino acids 247-467 ofSEQ ID NO:5) of human 33408 with a consensus amino acid sequence (SEQ IDNO:9) derived from a hidden Markov model is depicted in FIG. 4A. Analignment of the ion transport protein domain (amino acids 198-383 ofSEQ ID NO:8) of human 12189 with a consensus amino acid sequence (SEQ IDNO:9) derived from a hidden Markov model is depicted in FIG. 6B.

In a preferred embodiment, a 52906, 33408, or 12189 polypeptide orprotein has an “ion transport protein domain” or a region which includesat least about 150 to 280 more preferably about 170 to 260 or 180 to 230amino acid residues and has at least about 50%, 60%, 70% 80% 90% 95%,99%, or 100% homology with a “ion transport protein domain,” e.g., theion transport protein domain of human 52906, 33408, or 12189 (e.g.,residues 472-661 of SEQ ID NO:2, 247-467 of SEQ ID NO:5, or 198-383 ofSEQ ID NO:8).

A 33408 molecule can further include a cyclic nucleotide binding domainor regions homologous with a “cyclic nucleotide binding domain.”

As used herein, the term “cyclic nucleotide binding domain” includes anamino acid sequence of about 40-180 amino acid residues in length andhaving a bit score for the alignment of the sequence to the cyclicnucleotide binding domain (HMM) of at least 50. Preferably, a cyclicnucleotide binding domain is capable of binding a cyclic nucleotide.Preferably, a cyclic nucleotide binding domain includes at least about50-150 amino acids, more preferably about 70-120 amino acid residues, orabout 80-100 amino acids and has a bit score for the alignment of thesequence to the cyclic nucleotide binding domain (HMM) of at least 80 orgreater. The cyclic nucleotide binding domain (HMM) has been assignedthe PFAM Accession PF00027 (httD://genome.wustl.edu/Pfam/html). Analignment of the cyclic nucleotide binding domain (amino acids 565 to655 of SEQ ID NO:5) of human 33408 with a consensus amino acid sequence(SEQ ID NO:10) derived from a hidden Markov model is depicted in FIG.4B.

In a preferred embodiment a 33408 polypeptide or protein has a “cyclicnucleotide binding domain” or a region which includes at least about50-150, more preferably about 70-120 or 80-100 amino acid residues andhas at least about 50%, 60%, 70% 80% 90% 95%, 99%, or 100% homology witha “cyclic nucleotide binding domain,” e.g., the cyclic nucleotidebinding domain of human 33408 (e.g., residues 565 to 655 of SEQ IDNO:5).

A 12189 polypeptide can further include a “potassium channeltetramerisation domain” or regions homologous with a “potassium channeltetramerisation domain.”

As used herein, the term “potassium channel tetramerisation domain”includes an amino acid sequence of about 50 to 200 amino acid residuesin length and having a bit score for the alignment of the sequence tothe potassium channel tetramerisation domain profile (Pfam HMM) of atleast 100. A “potassium channel tetramerisation domain” promotes theassembly of alpha-subunits into functional tetrameric channels.Preferably, a potassium channel tetramerisation domain includes at leastabout 60 to 150 amino acids, more preferably about 70 to 130 amino acidresidues, or about 90 to 110 amino acids and has a bit score for thealignment of the sequence to the potassium channel tetramerisationdomain (HMM) of at least 165 or greater. The potassium channeltetramerisation domain (HMM) has been assigned the PFAM Accession NumberPF02214 (http;//genome.wustl.edu/Pfam/.html). An alignment of thepotassium channel tetramerisation domain (amino acids 3-101 of SEQ IDNO:8) of human 12189 with a consensus amino acid sequence (SEQ ID NO:11)derived from a hidden Markov model is depicted in FIG. 6A.

In a preferred embodiment, a 12189 polypeptide or protein has a“potassium channel tetramerisation domain” or a region which includes atleast about 60 to 150 more preferably about 70 to 130 or 90 to 110 aminoacid residues and has at least about 50%, 60%, 70% 80% 90% 95%, 99%, or100% homology with a “potassium channel tetramerisation domain,” e.g.,the potassium channel tetramerisation domain of human 12189 (e.g.,residues 3-101 of SEQ ID NO:8).

To identify the presence of an “ion transport protein” domain, a “cyclicnucleotide-binding” domain, or a “potassium channel tetramerisation”domain in a 52906, 33408, or 12189 protein sequence, and make thedetermination that a polypeptide or protein of interest has a particularprofile, the amino acid sequence of the protein can be searched againstthe Pfam database of HMMs (e.g., the Pfam database, release 2.1) usingthe default parameters(http://www.sanger.ac.uk/Software/Pfam/HMM_search). For example, thehmmsf program, which is available as part of the HMMER package of searchprograms, is a family specific default program for MILPAT0063 and ascore of 15 is the default threshold score for determining a hit.Alternatively, the threshold score for determining a hit can be lowered(e.g., to 8 bits). A description of the Pfam database can be found inSonhammer et al. (1997) Proteins 28(3):405-420 and a detaileddescription of HMMs can be found, for example, in Gribskov et al. (1990)Meth. Enzymol. 183:146-159; Gribskov et al.(1987) Proc. Natl. Acad. Sci.USA 84:4355-4358; Krogh et al. (1994) J. Mol. Biol. 235:1501-1531; andStultz et al. (1993) Protein Sci. 2:305-314, the contents of which areincorporated herein by reference.

A 33408 polypeptide can further include a “PAS domain” or regionshomologous with a “PAS domain”. As used herein, a “PAS domain” includesan amino acid sequence of about 100-200 amino acid residues in lengththat is involved in ligand and/or protein-protein interactions.Preferably, the PAS domain interacts with the body of the channel,affecting gating, inactivation, and/or voltage sensitivity. Preferably,the PAS domain is located at the N-terminal cytoplasmic region of the33408 polypeptide.

In a preferred embodiment, a 33408 polypeptide or protein has a “PASdomain” or a region which includes at least about 50-220, morepreferably about 100-200 or 120-140 amino acid residues and has at leastabout 50%, 60%, 70% 80% 90% 95%, 99%, or 100% homology with a “PASdomain,” e.g., the PAS domain of human 33408 (e.g., residues 1-134 ofSEQ ID NO:5).

A 33408. polypeptide can further include a “PAC domain” or regionshomologous with a “PAC domain”. As used herein, a “PAC domain” includesan amino acid sequence of about 30-50 amino acid residues in length.Preferably, the PAC domain contributes to the folding of the PAS domain.Preferably, the PAC domain is located at the C-terminal end of the PASdomain in a 33408 polypeptide.

In a preferred embodiment, a 33408 polypeptide or protein has a “PACdomain” or a region which includes at least about 20-70 or 30-50 aminoacid residues and has at least about 50%, 60%, 70% 80% 90% 95%, 99%, or100% homology with a “PAC domain,” e.g., the PAC domain of human 33408(e.g., residues 92-132 of SEQ ID NO:5).

A 52906 family member can include at least one (preferably two, three,four, five, or six) transmembrane domain, at least one (preferably twoor three) cytoplasmic domain, at least one (preferably two or three)extracellular domain, at least one P-loop domain, and at least one iontransport protein domain. Furthermore, a 52906, 33408, or 12189 familymember can include: at least one, two, three, four, five, and preferablysix predicted N-glycosylation sites (PS00001); at least one predictedglycosaminoglycan attachment site (PS00002); at least one, two, three,and preferably four predicted cAMP- and cGMP-dependent protein kinasephosphorylation sites (PS00004); at least one, two, three, four, five,six, seven, eight, nine, 10, 11, 12, and preferably 13 predicted ProteinKinase C phosphorylation sites (PS00005); at least one, two, three,four, five, six, and preferably seven predicted Casein Kinase IIphosphorylation sites (PS00006); at least one, two, three, four, five,six, seven, eight, nine, 10, 11, 12, 13, 14, and preferably 15 predictedN-myristoylation sites (PS00008); and at least one predicted coiled coildomain.

A 33408 family member can include at least one (preferably two, three,four, five, or six) transmembrane domain, at least one (preferably twoor three) cytoplasmic domain, at least one (preferably two or three)extracellular domain, at least one P-loop domain, and at least one iontransport protein domain. A 33408 family member can further include acyclic nucleotide-binding domain. A 33408 family member can furtherinclude a PAS domain and a PAC domain. Furthermore, a 33408 familymember can include: at least one, two, three, four, five, six, andpreferably seven predicted N-glycosylation sites (PS00001); at least oneand preferably two predicted cAMP- and cGMP-dependent protein kinasephosphorylation sites (PS00004); at least one, two, three, four, five,six, seven, eight, nine, 10, 11, 12, and preferably 13 predicted ProteinKinase C phosphorylation sites (PS00005); at least one, two, three,four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, andpreferably 16 predicted Casein Kinase II phosphorylation sites(PS00006); at least one, two, three, four, five, six, seven, andpreferably eight predicted N-myristoylation sites (PS00008); at leastone predicted amidation site (PS00009); at least one predicted leucinezipper pattern (PS00029); and at least one predicted coiled coil domain.

A 12189 family member can include at least one (preferably two, three,four, five, or six) transmembrane domain, at least one (preferably twoor three) cytoplasmic domain, at least one (preferably two or three)extracellular domain, at least one P-loop domain, and at least one iontransport protein domain. A 12189 family member can further include apotassium channel tetramerisation domain. Furthermore, a 12189 familymember can include: at least one and preferably two predictedN-glycosylation sites (PS00001); at least one and preferably twopredicted Protein Kinase C phosphorylation sites (PS00005); at leastone, two, three, four, and preferably five predicted Casein Kinase IIphosphorylation sites (PS00006); at least one predicted tyrosine kinasephosphorylation site (PS00007); at least one, two, three, four, andpreferably five predicted N-myristoylation sites (PS00008); and at leastone predicted leucine zipper pattern (PS00029).

As the 52906, 33408, or 12189 polypeptides of the invention may modulate52906, 33408, or 12189-mediated activities, e.g., potassium channelmediated activities, they may be useful as of for developing noveldiagnostic and therapeutic agents for 52906, 33408, or 12189-mediated orrelated disorders, e.g., potassium channel associated disorders, asdescribed below.

As used herein, a “52906, 33408, or 12189 activity”, “biologicalactivity of 52906, 33408, or 12189” or “functional activity of 52906,33408, or 12189”, refers to an activity exerted by a 52906, 33408, or12189 protein, polypeptide or nucleic acid molecule. For example, a52906, 33408, or 12189 activity can be an activity exerted by 52906,33408, or 12189 in a physiological milieu on, e.g., a 52906, 33408, or12189-responsive cell or on a 52906, 33408, or 12189 substrate, e.g., aprotein substrate. A 52906, 33408, or 12189 activity can be determinedin vivo or in vitro. In one embodiment, a 52906, 33408, or 12189activity is a direct activity, such as an association with a 52906,33408, or 12189 target molecule. A “target molecule” or “bindingpartner” is a molecule with which a 52906, 33408, or 12189 protein bindsor interacts in nature. In an exemplary embodiment, 52906, 33408, or12189 is an ion channel, e.g., a potassium channel.

A 52906, 33408, or 12189 activity can also be an indirect activity,e.g., a cellular signaling activity mediated by interaction of the52906, 33408, or 12189 protein with a 52906, 33408, or 12189 ligand,e.g., a potassium ion. The features of the 52906, 33408, or 12189molecules of the present invention can provide similar biologicalactivities as potassium channel family members. For example, the 52906,33408, or 12189 proteins of the present invention can have one or moreof the following activities: (1) interacting with a non-52906, 33408, or12189 protein molecule; (2) activating a 52906, 33408, or12189-dependent signal transduction pathway; (3) modulating the releaseof neurotransmitters; (4) modulating membrane excitability; (5)influencing the resting potential of membranes, wave forms andfrequencies of action potentials, and thresholds of excitation; (6)binding a cyclic nucleotide; (7) contributing to the formation ofpotassium channels; (8) contributing to the formation ofcalcium-activated, voltage independent potassium channels; (9)modulating repolarization of the neuronal cell membrane; (10)contributing to the formation of voltage-gated potassium channels; (11)contributing to the formation of cyclic nucleotide-gated potassiumchannels; (12) modulating the flow of K⁺ ions through a cell membrane;and (13) modulating processes which underlie learning and memory, suchas integration of sub-threshold synaptic responses and the conductanceof back-propagating action potentials.

Based on the above-described sequence similarities, the 52906, 33408, or12189 molecules of the present invention are predicted to have similarbiological activities as potassium channel family members. In addition,52906 and 33408 mRNA was found to be highly expressed in cells derivedfrom brain and heart (see Tables 3 and 4). Thus, the 52906, 33408, or12189 molecules can act as novel diagnostic targets and therapeuticagents for controlling potassium channel associated disorders. Examplesof such disorders include neurological disorders and cardiac-relateddisorders.

As used herein, a “potassium channel associated disorder” includes adisorder, disease or condition which is characterized by a misregulationof a potassium channel mediated activity. Potassium channel associateddisorders can detrimentally affect conveyance of sensory impulses fromthe periphery to the brain and/or conductance of motor impulses from thebrain to the periphery; integration of reflexes; interpretation ofsensory impulses; cellular proliferation, growth, differentiation, ormigration, and emotional, intellectual (e.g.,. learning and memory), ormotor processes. Examples of potassium channel associated disordersinclude CNS disorders such as cognitive and neurodegenerative disorders,examples of which include, but are not limited to, Alzheimer's disease,dementias related to Alzheimer's disease (such as Pick's disease),Parkinson's and other Lewy diffuse body diseases, senile dementia,Huntington's disease, Gilles de la Tourette's syndrome, multiplesclerosis, amyotrophic lateral sclerosis, progressive supranuclearpalsy, epilepsy, and Jakob-Creutzfieldt disease; autonomic functiondisorders such as hypertension and sleep disorders, and neuropsychiatricdisorders, such as depression, schizophrenia, schizoaffective disorder,korsakoff's psychosis, mania, anxiety disorders, or phobic disorders;learning or memory disorders, e.g., amnesia or age-related memory loss,attention deficit disorder, dysthymic disorder, major depressivedisorder, mania, obsessive-compulsive disorder, psychoactive substanceuse disorders, anxiety, phobias, panic disorder, as well as bipolaraffective disorder, e.g., severe bipolar affective (mood) disorder(BP-1), and bipolar affective neurological disorders, e.g., migraine andobesity. Further CNS-related disorders include, for example, thoselisted in the American Psychiatric Association's Diagnostic andStatistical manual of Mental Disorders (DSM), the most current versionof which is incorporated herein by reference in its entirety.

Further examples of potassium channel associated disorders includecardiac-related disorders. Cardiovascular system disorders in which the52906, 33408, or 12189 molecules of the invention may be directly orindirectly involved include arteriosclerosis, ischemia reperfusioninjury, restenosis, arterial inflammation, vascular wall remodeling,ventricular remodeling, rapid ventricular pacing, coronarymicroembolism, tachycardia, bradycardia, pressure overload, aorticbending, coronary artery ligation, vascular heart disease, atrialfibrilation, Jervell syndrome, Lange-Nielsen syndrome, long-QT syndrome,congestive heart failure, sinus node dysfunction, angina, heart failure,hypertension, atrial fibrillation, atrial flutter, dilatedcardiomyopathy, idiopathic cardiomyopathy, myocardial infarction,coronary artery disease, coronary artery spasm, and arrhythmia. 52906,33408, or 12189-mediated or related disorders also include disorders ofthe musculoskeletal system such as paralysis and muscle weakness, e.g.,ataxia, myotonia, and myokymia.

As used herein, a “potassium channel mediated activity” includes anactivity which involves a potassium channel, e.g., a potassium channelin a neuronal cell, a muscle cell, or a thymus cell associated withreceiving, conducting, and transmitting signals in, for example, thenervous system. Potassium channel mediated activities include release ofneurotransmitters, e.g., dopamine or norepinephrine, from cells, e.g.,neuronal cells; modulation of resting potential of membranes, wave formsand frequencies of action potentials, and thresholds of excitation;participation in signal transduction pathways, and modulation ofprocesses such as integration of sub-threshold synaptic responses andthe conductance of back-propagating action potentials in, for example,neuronal cells or muscle cells.

The presence of 52906, 33408, or 12189 RNA or protein can be used toidentify a cell or tissue, or other biological sample, as being derivedfrom the brain, e.g., cerebral cortex, from the heart, from a muscle, orof neuronal origin. Expression can be determined by evaluating RNA,e.g., by hybridization of a 52906, 33408, or 12189 specific probe, orwith a 52906, 33408, or 12189 specific antibody.

The 52906, 33408, or 12189 protein, fragments thereof, and derivativesand other variants of the sequence in SEQ ID NO:2, SEQ ID NO:5, or SEQID NO:8 thereof are collectively referred to as “polypeptides orproteins of the invention” or “52906, 33408, or 12189 polypeptides orproteins”. Nucleic acid molecules encoding such polypeptides or proteinsare collectively referred to as “nucleic acids of the invention” or“52906, 33408, or 12189 nucleic acids.” 52906, 33408, or 12189 moleculesrefer to 52906, 33408, or 12189 nucleic acids, polypeptides, andantibodies.

As used herein, the term “nucleic acid molecule” includes DNA molecules(e.g., a cDNA or genomic DNA), RNA molecules (e.g., an mRNA) and analogsof the DNA or RNA. A DNA or RNA analog can be synthesized fromnucleotide analogs. The nucleic acid molecule can be single-stranded ordouble-stranded, but preferably is double-stranded DNA.

The term “isolated nucleic acid molecule” or “purified nucleic acidmolecule” includes nucleic acid molecules that are separated from othernucleic acid molecules present in the natural source of the nucleicacid. For example, with regards to genomic DNA, the term “isolated”includes nucleic acid molecules which are separated from the chromosomewith which the genomic DNA is naturally associated. Preferably, an“isolated” nucleic acid is free of sequences which naturally flank thenucleic acid (i.e., sequences located at the 5′ and/or 3′ ends of thenucleic acid) in the genomic DNA of the organism from which the nucleicacid is derived. For example, in various embodiments, the isolatednucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2kb, 1 kb, 0.5 kb or 0.1 kb of 5′ and/or 3′ nucleotide sequences whichnaturally flank the nucleic acid molecule in genomic DNA of the cellfrom which the nucleic acid is derived. Moreover, an “isolated” nucleicacid molecule, such as a cDNA molecule, can be substantially free ofother cellular material, or culture medium when produced by recombinanttechniques, or substantially free of chemical precursors or otherchemicals when chemically synthesized.

As used herein, the term “hybridizes under low stringency, mediumstringency, high stringency, or very high stringency conditions”describes conditions for hybridization and washing. Guidance forperforming hybridization reactions can be found in Current Protocols inMolecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6, which isincorporated by reference. Aqueous and nonaqueous methods are describedin that reference and either can be used. Specific hybridizationconditions referred to herein are as follows: 1) low stringencyhybridization conditions in 6×sodium chloride/sodium citrate (SSC) atabout 45° C., followed by two washes in 0.2×SSC, 0.1% SDS at least at50° C. (the temperature of the washes can be increased to 55° C. for lowstringency conditions); 2) medium stringency hybridization conditions in6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1%SDS at 60° C.; 3) high stringency hybridization conditions in 6×SSC atabout 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 65°C.; and preferably 4) very high stringency hybridization conditions are0.5M sodium phosphate, 7% SDS at 65° C., followed by one or more washesat 0.2×SSC, 1% SDS at 65° C. Very high stringency conditions (4) are thepreferred conditions and the ones that should be used unless otherwisespecified.

Preferably, an isolated nucleic acid molecule of the invention thathybridizes under a stringency condition described herein to the sequenceof SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:7,corresponds to a naturally-occurring nucleic acid molecule.

As used herein, a “naturally-occurring” nucleic acid molecule refers toan RNA or DNA molecule having a nucleotide sequence that occurs innature. For example a naturally occurring nucleic acid molecule canencode a natural protein. As used herein, the terms “gene” and“recombinant gene” refer to nucleic acid molecules which include atleast an open reading frame encoding a 52906, 33408, or 12189 protein.The gene can optionally further include non-coding sequences, e.g.,regulatory sequences and introns. Preferably, a gene encodes a mammalian52906, 33408, or 12189 protein or derivative thereof.

An “isolated” or “purified” polypeptide or protein is substantially freeof cellular material or other contaminating proteins from the cell ortissue source from which the protein is derived, or substantially freefrom chemical precursors or other chemicals when chemically synthesized.“Substantially free” means that a preparation of 52906, 33408, or 12189protein is at least 10% pure. In a preferred embodiment, the preparationof 52906, 33408, or 12189 protein has less than about 30%, 20%, 10% andmore preferably 5% (by dry weight), of non-52906, 33408, or 12189protein (also referred to herein as a “contaminating protein”), or ofchemical precursors or non-52906, 33408, or 12189 chemicals. When the52906, 33408, or 12189 protein or biologically active portion thereof isrecombinantly produced, it is also preferably substantially free ofculture medium, i.e., culture medium represents less than about 20%,more preferably less than about 10%, and most preferably less than about5% of the volume of the protein preparation. The invention includesisolated or purified preparations of at least 0.01, 0.1, 1.0, and 10milligrams in dry weight.

A “non-essential” amino acid residue is a residue that can be alteredfrom the wild-type sequence of 52906, 33408, or 12189 without abolishingor substantially altering a 52906, 33408, or 12189 activity. Preferablythe alteration does not substantially alter the 52906, 33408, or 12189activity, e.g., the activity is at least 20%, 40%, 60%, 70% or 80% ofwild-type. An “essential” amino acid residue is a residue that, whenaltered from the wild-type sequence of 52906, 33408, or 12189, resultsin abolishing a 52906, 33408, or 12189 activity such that less than 20%of the wild-type activity is present. For example, conserved amino acidresidues in 52906, 33408, or 12189 are predicted to be particularlyunamenable to alteration.

A “conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, apredicted nonessential amino acid residue in a 52906, 33408, or 12189protein is preferably replaced with another amino acid residue from thesame side chain family. Alternatively, in another embodiment, mutationscan be introduced randomly along all or part of a 52906, 33408, or 12189coding sequence, such as by saturation mutagenesis, and the resultantmutants can be screened for 52906, 33408, or 12189 biological activityto identify mutants that retain activity. Following mutagenesis of SEQID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:7, theencoded protein can be expressed recombinantly and the activity of theprotein can be determined.

As used herein, a “biologically active portion” of a 52906, 33408, or12189 protein includes a fragment of a 52906, 33408, or 12189 proteinwhich participates in an interaction, e.g., an intramolecular or aninter-molecular interaction. An inter-molecular interaction can be aspecific binding interaction or an enzymatic interaction (e.g., theinteraction can be transient and a covalent bond is formed or broken).An inter-molecular interaction can be between a 52906, 33408, or 12189molecule and a non-52906, 33408, or 12189 molecule or between a first52906, 33408, or 12189 molecule and a second 52906, 33408, or 12189molecule (e.g., a dimerization interaction). Biologically activeportions of a 52906, 33408, or 12189 protein include peptides comprisingamino acid sequences sufficiently homologous to or derived from theamino acid sequence of the 52906, 33408, or 12189 protein, e.g., theamino acid sequence shown in SEQ ID NO:2, SEQ ID NO:5, or SEQ ID NO:8,which include less amino acids than the full length 52906, 33408, or12189 proteins, and exhibit at least one activity of a 52906, 33408, or12189 protein. Typically, biologically active portions comprise a domainor motif with at least one activity of the 52906, 33408, or 12189protein, e.g., the ability to modulate the flow of K⁺ ions through acell membrane and/or the ability to modulate the transmission of signalsin an electrically excitable cell, e.g., a neuronal cell or a musclecell. A biologically active portion of a 52906, 33408, or 12189 proteincan be a polypeptide which is, for example, 10, 25, 50, 100, 200 or moreamino acids in length. Biologically active portions of a 52906, 33408,or 12189 protein can be used as targets for developing agents whichmodulate a 52906, 33408, or 12189 mediated activity, e.g., the abilityto modulate the flow of K⁺ ions through a cell membrane and/or theability to modulate the transmission of signals in an electricallyexcitable cell, e.g., a neuronal cell or a muscle cell.

Calculations of homology or sequence identity between sequences (theterms are used interchangeably herein) are performed as follows.

To determine the percent identity of two amino acid sequences, or of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes). Ina preferred embodiment, the length of a reference sequence aligned forcomparison purposes is at least 30%, preferably at least 40%, morepreferably at least 50%, 60%, and even more preferably at least 70%,80%, 90%, 100% of the length of the reference sequence. The amino acidresidues or nucleotides at corresponding amino acid positions ornucleotide positions are then compared. When a position in the firstsequence is occupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position (as used herein amino acid or nucleic acid“identity” is equivalent to amino acid or nucleic acid “homology”).

The percent identity between the two sequences is a function of thenumber of identical positions shared by the sequences, taking intoaccount the number of gaps, and the length of each gap, which need to beintroduced for optimal alignment of the two sequences.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In a preferred embodiment, the percent identity between twoamino acid sequences is determined using the Needleman and Wunsch((1970) J. Mol. Biol. 48:444-453 ) algorithm which has been incorporatedinto the GAP program in the GCG software package (available athttp://www.gcg.com), using either a Blossum 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, thepercent identity between two nucleotide sequences is determined usingthe GAP program in the GCG software package (available athttp://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. Aparticularly preferred set of parameters (and the one that should beused unless otherwise specified) are a Blossum 62 scoring matrix with agap penalty of 12, a gap extend penalty of 4, and a frameshift gappenalty of 5.

The percent identity between two amino acid or nucleotide sequences canbe determined using the algorithm of E. Meyers and W. Miller ((1989)CABIOS, 4:11-17) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4.

The nucleic acid and protein sequences described herein can be used as a“query sequence” to perform a search against public databases to, forexample, identify other family members or related sequences. Suchsearches can be performed using the NBLAST and XBLAST programs (version2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLASTnucleotide searches can be performed with the NBLAST program, score=100,wordlength=12 to obtain nucleotide sequences homologous to 52906, 33408,or 12189 nucleic acid molecules of the invention. BLAST protein searchescan be performed with the XBLAST program, score=50, wordlength=3 toobtain amino acid sequences homologous to 52906, 33408, or 12189 proteinmolecules of the invention. To obtain gapped alignments for comparisonpurposes, Gapped BLAST can be utilized as described in Altschul et al.,(1997) Nucleic Acids Res. 25:3389-3402. When utilizing BLAST and GappedBLAST programs, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.

Particularly preferred 52906, 33408, or 12189 polypeptides of thepresent invention have an amino acid sequence substantially identical tothe amino acid sequence of SEQ ID NO:2, SEQ ID NO:5, or SEQ ID NO:8. Inthe context of an amino acid sequence, the term “substantiallyidentical” is used herein to refer to a first amino acid that contains asufficient or minimum number of amino acid residues that are i)identical to, or ii) conservative substitutions of aligned amino acidresidues in a second amino acid sequence such that the first and secondamino acid sequences can have a common structural domain and/or commonfunctional activity. For example, amino acid sequences that contain acommon structural domain having at least about 60%, or 65% identity,likely 75% identity, more likely 85%, 90%. 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% identity to SEQ ID NO:2, SEQ ID NO:5, or SEQ ID NO:8 aretermed substantially identical.

In the context of nucleotide sequence, the term “substantiallyidentical” is used herein to refer to a first nucleic acid sequence thatcontains a sufficient or minimum number of nucleotides that areidentical to aligned nucleotides in a second nucleic acid sequence suchthat the first and second nucleotide sequences encode a polypeptidehaving common functional activity, or encode a common structuralpolypeptide domain or a common functional polypeptide activity. Forexample, nucleotide sequences having at least about 60%, or 65%identity, likely 75% identity, more likely 85%, 90%. 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:6, or SEQ ID NO:7 are termed substantially identical.

“Misexpression or aberrant expression”, as used herein, refers to anon-wildtype pattern of gene expression at the RNA or protein level. Itincludes: expression at non-wild type levels, i.e., over- orunder-expression; a pattern of expression that differs from wild type interms of the time or stage at which the gene is expressed, e.g.,increased or decreased expression (as compared with wild type) at apredetermined developmental period or stage; a pattern of expressionthat differs from wild type in terms of altered, e.g., increased ordecreased, expression (as compared with wild type) in a predeterminedcell type or tissue type; a pattern of expression that differs from wildtype in terms of the splicing size, translated amino acid sequence,post-transitional modification, or biological activity of the expressedpolypeptide; a pattern of expression that differs from wild type interms of the effect of an environmental stimulus or extracellularstimulus on expression of the gene, e.g., a pattern of increased ordecreased expression (as compared with wild type) in the presence of anincrease or decrease in the strength of the stimulus.

“Subject,” as used herein, refers to human and non-human animals. Theterm “non-human animals” of the invention includes all vertebrates,e.g., mammals, such as non-human primates (particularly higherprimates), sheep, dog, rodent (e.g., mouse or rat), guinea pig, goat,pig, cat, rabbits, cow, and non-mammals, such as chickens, amphibians,reptiles, etc. In a preferred embodiment, the subject is a human. Inanother embodiment, the subject is an experimental animal or animalsuitable as a disease model.

A “purified preparation of cells”, as used herein, refers to an in vitropreparation of cells. In the case cells from multicellular organisms(e.g., plants and animals), a purified preparation of cells is a subsetof cells obtained from the organism, not the entire intact organism. Inthe case of unicellular microorganisms (e.g., cultured cells andmicrobial cells), it consists of a preparation of at least 10% and morepreferably 50% of the subject cells.

Various aspects of the invention are described in further detail below.

Isolated Nucleic Acid Molecules

In one aspect, the invention provides, an isolated or purified, nucleicacid molecule that encodes a 52906, 33408, or 12189 polypeptidedescribed herein, e.g., a full-length 52906, 33408, or 12189 protein ora fragment thereof, e.g., a biologically active portion of 52906, 33408,or 12189 protein. Also included is a nucleic acid fragment suitable foruse as a hybridization probe, which can be used, e.g., to identify anucleic acid molecule encoding a polypeptide of the invention, 52906,33408, or 12189 mRNA, and fragments suitable for use as primers, e.g.,PCR primers for the amplification or mutation of nucleic acid molecules.

In one embodiment, an isolated nucleic acid molecule of the inventionincludes the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:4, orSEQ ID NO:7, or a portion of any of these nucleotide sequences. In oneembodiment, the nucleic acid molecule includes sequences encoding thehuman 52906, 33408, or 12189 protein (i.e., “the coding region” of SEQID NO:1, as shown in SEQ ID NO:3 or “the coding region” of SEQ ID NO:4,as shown in SEQ ID NO:6), as well as 5′ untranslated sequences.Alternatively, the nucleic acid molecule can include only the codingregion of SEQ ID NO:1, SEQ ID NO:4, or SEQ ID NO:7 (e.g., SEQ ID NO:3 orSEQ ID NO:6) and, e.g., no flanking sequences which normally accompanythe subject sequence. In another embodiment, the nucleic acid moleculeencodes a sequence corresponding to a fragment of the protein from aboutamino acids 472-661 of SEQ ID NO:2, amino acids 247-467 of SEQ ID NO:5,amino acids 565-655 of SEQ ID NO:5, amino acids 3-101 of SEQ ID NO:8, oramino acids 198-383 of SEQ ID NO:8.

In another embodiment, an isolated nucleic acid molecule of theinvention includes a nucleic acid molecule which is a complement of thenucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQID NO:6, or SEQ ID NO:7, or a portion of any of these nucleotidesequences. In other embodiments, the nucleic acid molecule of theinvention is sufficiently complementary to the nucleotide sequence shownin SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:7,such that it can hybridize (e.g., under a stringency condition describedherein) to the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3,SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:7, thereby forming a stableduplex.

In one embodiment, an isolated nucleic acid molecule of the presentinvention includes a nucleotide sequence which is at least about: 60%,65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or more homologous to the entire length of the nucleotide sequenceshown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, or SEQ IDNO:7, or a portion, preferably of the same length, of any of thesenucleotide sequences.

52906, 33408, or 12189 Nucleic Acid Fragments

A nucleic acid molecule of the invention can include only a portion ofthe nucleic acid sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQID NO:6, or SEQ ID NO:7. For example, such a nucleic acid molecule caninclude a fragment which can be used as a probe or primer or a fragmentencoding a portion of a 52906, 33408, or 12189 protein, e.g., animmunogenic or biologically active portion of a 52906, 33408, or 12189protein. A fragment can comprise those nucleotides of SEQ ID NO:1, SEQID NO:3, SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:7, which encode an iontransport protein domain of human 52906, 33408, or 12189. The nucleotidesequence determined from the cloning of the 52906, 33408, or 12189 geneallows for the generation of probes and primers designed for use inidentifying and/or cloning other 52906, 33408, or 12189 family members,or fragments thereof, as well as 52906, 33408, or 12189 homologues, orfragments thereof, from other species.

In another embodiment, a nucleic acid includes a nucleotide sequencethat includes part, or all, of the coding region and extends into either(or both) the 5′ or 3′ noncoding region. Other embodiments include afragment which includes a nucleotide sequence encoding an amino acidfragment described herein. Nucleic acid fragments can encode a specificdomain or site described herein (e.g., an ion transport protein domain,a cyclic nucleotide-binding domain, a potassium channel tetramerisationdomain, a transmembrane domain, a cytoplasmic domain, an extracellulardomain, a Pore-loop domain, or a PAS domain) or fragments thereof,particularly fragments thereof which are at least 100, 200, 300, 400, or500 amino acids in length. Fragments also include nucleic acid sequencescorresponding to specific amino acid sequences described above orfragments thereof. Nucleic acid fragments should not to be construed asencompassing those fragments that may have been disclosed prior to theinvention.

A nucleic acid fragment can include a sequence corresponding to adomain, region, or functional site described herein. A nucleic acidfragment can also include one or more domain, region, or functional sitedescribed herein. Thus, for example, a 52906, 33408, or 12189 nucleicacid fragment can include a sequence corresponding to an ion transportprotein domain, a cyclic nucleotide-binding domain, a potassium channeltetramerisation domain, a transmembrane domain, a cytoplasmic domain, anextracellular domain, a Pore-loop domain, or a PAS domain.

52906, 33408, or 12189 probes and primers are provided. Typically aprobe/primer is an isolated or purified oligonucleotide. Theoligonucleotide typically includes a region of nucleotide sequence thathybridizes under a stringency condition described herein to at leastabout 7, 12 or 15, preferably about 20 or 25, more preferably about 30,35, 40, 45, 50, 55, 60, 65, or 75 consecutive nucleotides of a sense orantisense sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:6, or SEQ ID NO:7, or of a naturally occurring allelic variant ormutant of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, or SEQ IDNO:7.

In a preferred embodiment the nucleic acid is a probe which is at least5 or 10, and less than 200, more preferably less than 100, or less than50, base pairs in length. It should be identical, or differ by 1, orless than in 5 or 10 bases, from a sequence disclosed herein. Ifalignment is needed for this comparison the sequences should be alignedfor maximum homology. “Looped” out sequences from deletions orinsertions, or mismatches, are considered differences.

A probe or primer can be derived from the sense or anti-sense strand ofa nucleic acid which encodes: an ion transport protein domain, a cyclicnucleotide-binding domain, a potassium channel tetramerisation domain, atransmembrane domain, a cytoplasmic domain, an extracellular domain, aPore-loop domain, or a PAS domain. The locations of these domains in SEQID NO:2, SEQ ID NO:5, and SEQ ID NO:8 are described in Table 2.

In another embodiment a set of primers is provided, e.g., primerssuitable for use in a PCR, which can be used to amplify a selectedregion of a 52906, 33408, or 12189 sequence, e.g., a domain, region,site or other sequence described herein. The primers should be at least5, 10, or 50 base pairs in length and less than 100, or less than 200,base pairs in length. The primers should be identical, or differs by onebase from a sequence disclosed herein or from a naturally occurringvariant. For example, primers suitable for amplifying all or a portionof any of the following regions are provided: an ion transport proteindomain, a cyclic nucleotide-binding domain, a potassium channeltetramerisation domain, a transmembrane domain, a cytoplasmic domain, anextracellular domain, a Pore-loop domain, or a PAS domain.

A nucleic acid fragment can encode an epitope bearing region of apolypeptide described herein.

A nucleic acid fragment encoding a “biologically active portion of a52906, 33408, or 12189 polypeptide” can be prepared by isolating aportion of the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:6, or SEQ ID NO:7, which encodes a polypeptide having a52906, 33408, or 12189 biological activity (e.g., the biologicalactivities of the 52906, 33408, or 12189 proteins are described herein),expressing the encoded portion of the 52906, 33408, or 12189 protein(e.g., by recombinant expression in vitro) and assessing the activity ofthe encoded portion of the 52906, 33408, or 12189 protein. For example,a nucleic acid fragment encoding a biologically active portion of 52906,33408, or 12189 includes ion transport protein domain, e.g., amino acids472-661 of SEQ ID NO:2, amino acids 247-467 of SEQ ID NO:5, or aminoacids 198-383 of SEQ ID NO:8. A nucleic acid fragment encoding abiologically active portion of a 52906, 33408, or 12189 polypeptide, maycomprise a nucleotide sequence which is greater than 300 or morenucleotides in length.

In preferred embodiments, a nucleic acid includes a nucleotide sequencewhich is about 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200,1300, 1400, 1500, 2000, 2500, 3000, 3300, 3400, 3500, or morenucleotides in length and hybridizes under a stringency conditiondescribed herein to a nucleic acid molecule of SEQ ID NO:1, SEQ ID NO:3,SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:7.

In preferred embodiments, the fragment includes at least one, andpreferably at least 5, 10, 15, 25, 50, 100, 200, 300, 400, or 500nucleotides from nucleotides 1-2962, 3437-3525, 1-1441, 3182-3525, or1-2687 of SEQ ID NO:1.

In preferred embodiments, the fragment includes at least one, andpreferably at least 5, 10, 15, 25, 50, 75, 100, 200, 300, 500, 1000, or1500 nucleotides encoding a protein including 5, 10, 15, 20, 25, 30, 40,50, 100, 200, 300, 400, or 500 amino acids from amino acids 1-775,1-268, 1-683 of SEQ ID NO:2.

In preferred embodiments, the nucleic acid fragment includes anucleotide sequence that is other than the sequence of AA418096, V35457,Z51630, W63707, or W63702.

In preferred embodiments, the fragment comprises the coding region of52906, e.g., the nucleotide sequence of SEQ ID NO:3.

In preferred embodiments, the fragment includes at least one, andpreferably at least 5, 10, 15, 25, 50, 100, 200, 300, 400, or 500nucleotides from nucleotides 1-1844, 1-277, 1-252, or 3245-3553, of SEQID NO:4.

In preferred embodiments, the fragment includes the nucleotide sequenceof SEQ ID NO:6 and at least one, and preferably at least 5, 10, 15, 25,50, 75, 100, 200, 300, or 500 nucleotides, e.g., consecutivenucleotides, of SEQ ID NO:4.

In preferred embodiments, the fragment includes at least one, andpreferably at least 5, 10, 15, 25, 50, 75, 100, 200, 300, 500, 1000, or1500 nucleotides encoding a protein including 5, 10, 15, 20, 25, 30, 40,50, 100, 200, 300, 400, or 500 amino acids from amino acids 1-522 of SEQID NO:5.

In preferred embodiments, the nucleic acid fragment includes anucleotide sequence that is other than the sequence of U69185 or asequence described in WO01/04133 or WO01/29068.

In preferred embodiments, the fragment comprises the coding region of33408, e.g., the nucleotide sequence of SEQ ID NO:6.

52906, 33408, or 12189 Nucleic Acid Variants

The invention further encompasses nucleic acid molecules that differfrom the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:6, or SEQ ID NO:7. Such differences can be due todegeneracy of the genetic code (and result in a nucleic acid whichencodes the same 52906, 33408, or 12189 proteins as those encoded by thenucleotide sequence disclosed herein. In another embodiment, an isolatednucleic acid molecule of the invention has a nucleotide sequenceencoding a protein having an amino acid sequence which differs, by atleast 1, but less than 5, 10, 20, 50, or 100 amino acid residues thatshown in SEQ ID NO:2, SEQ ID NO:5, or SEQ ID NO:8. If alignment isneeded for this comparison the sequences should be aligned for maximumhomology. “Looped” out sequences from deletions or insertions, ormismatches, are considered differences.

Nucleic acids of the inventor can be chosen for having codons, which arepreferred, or non-preferred, for a particular expression system. E.g.,the nucleic acid can be one in which at least one codon, at preferablyat least 10%, or 20% of the codons has been altered such that thesequence is optimized for expression in E. coli, yeast, human, insect,or CHO cells.

Nucleic acid variants can be naturally occurring, such as allelicvariants (same locus), homologs (different locus), and orthologs(different organism) or can be non naturally occurring. Non-naturallyoccurring variants can be made by mutagenesis techniques, includingthose applied to polynucleotides, cells, or organisms. The variants cancontain nucleotide substitutions, deletions, inversions and insertions.Variation can occur in either or both the coding and non-coding regions.The variations can produce both conservative and non-conservative aminoacid substitutions (as compared in the encoded product).

In a preferred embodiment, the nucleic acid differs from that of SEQ IDNO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:7, e.g., asfollows: by at least one but less than 10, 20, 30, or 40 nucleotides; atleast one but less than 1%, 5%, 10% or 20% of the nucleotides in thesubject nucleic acid. If necessary for this analysis the sequencesshould be aligned for maximum homology. “Looped” out sequences fromdeletions or insertions, or mismatches, are considered differences.

Orthologs, homologs, and allelic variants can be identified usingmethods known in the art. These variants comprise a nucleotide sequenceencoding a polypeptide that is 50%, at least about 55%, typically atleast about 70-75%, more typically at least about 80-85%, and mosttypically at least about 90-95% or more identical to the nucleotidesequence shown in SEQ ID NO:2, SEQ ID NO:5, or SEQ ID NO:8 or a fragmentof this sequence. Such nucleic acid molecules can readily be identifiedas being able to hybridize under a stringency condition describedherein, to the nucleotide sequence shown in SEQ ID NO:2, SEQ ID NO:5, orSEQ ID NO:8 or a fragment of the sequence. Nucleic acid moleculescorresponding to orthologs, homologs, and allelic variants of the 52906,33408, or 12189 cDNAs of the invention can further be isolated bymapping to the same chromosome or locus as the 52906, 33408, or 12189gene.

Preferred variants include those that are correlated with the ability tomodulate the flow of K⁺ ions through a cell membrane and/or the abilityto modulate the transmission of signals in an electrically excitablecell, e.g., a neuronal cell or a muscle cell.

Allelic variants of 52906, 33408, or 12189, e.g., human 52906, 33408, or12189, include both functional and non-functional proteins. Functionalallelic variants are. naturally occurring amino acid sequence variantsof the 52906, 33408, or 12189 protein within a population that maintainthe ability to modulate the flow of K⁺ ions through a cell membraneand/or the ability to modulate the transmission of signals in anelectrically excitable cell, e.g., a neuronal cell or a muscle cell.Functional allelic variants will typically contain only conservativesubstitution of one or more amino acids of SEQ ID NO:2, SEQ ID NO:5, orSEQ ID NO:8, or substitution, deletion or insertion of non-criticalresidues in non-critical regions of the protein. Non-functional allelicvariants are naturally-occurring amino acid sequence variants of the52906, 33408, or 12189, e.g., human 52906, 33408, or 12189, proteinwithin a population that do not have the ability to modulate the flow ofK⁺ ions through a cell membrane and/or the ability to modulate thetransmission of signals in an electrically excitable cell, e.g., aneuronal cell or a muscle cell. Non-functional allelic variants willtypically contain a non-conservative substitution, a deletion, orinsertion, or premature truncation of the amino acid sequence of SEQ IDNO:2, SEQ ID NO:5, or SEQ ID NO:8, or a substitution, insertion, ordeletion in critical residues or critical regions of the protein.

Moreover, nucleic acid molecules encoding other 52906, 33408, or 12189family members and, thus, which have a nucleotide sequence which differsfrom the 52906, 33408, or 12189 sequences of SEQ ID NO:1, SEQ ID NO:3,SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:7 are intended to be within thescope of the invention.

Antisense Nucleic Acid Molecules, Ribozymes and Modified 52906, 33408,or 12189 Nucleic Acid Molecules

In another aspect, the invention features, an isolated nucleic acidmolecule which is antisense to 52906, 33408, or 12189. An “antisense”nucleic acid can include a nucleotide sequence which is complementary toa “sense” nucleic acid encoding a protein, e.g., complementary to thecoding strand of a double-stranded cDNA molecule or complementary to anmRNA sequence. The antisense nucleic acid can be complementary to anentire 52906, 33408, or 12189 coding strand, or to only a portionthereof (e.g., the coding region of human 52906, 33408, or 12189corresponding to SEQ ID NO:3 or SEQ ID NO:6). In another embodiment, theantisense nucleic acid molecule is antisense to a “noncoding region” ofthe coding strand of a nucleotide sequence encoding 52906, 33408, or12189 (e.g., the 5′ and 3′ untranslated regions).

An antisense nucleic acid can be designed such that it is complementaryto the entire coding region of 52906, 33408, or 12189 mRNA, but morepreferably is an oligonucleotide which is antisense to only a portion ofthe coding or noncoding region of 52906, 33408, or 12189 mRNA. Forexample, the antisense oligonucleotide can be complementary to theregion surrounding the translation start site of 52906, 33408, or 12189mRNA, e.g., between the −10 and +10 regions of the target genenucleotide sequence of interest. An antisense oligonucleotide can be,for example, about 7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, or more nucleotides in length.

An antisense nucleic acid of the invention can be constructed usingchemical synthesis and enzymatic ligation reactions using proceduresknown in the art. For example, an antisense nucleic acid (e.g., anantisense oligonucleotide) can be chemically synthesized using naturallyoccurring nucleotides or variously modified nucleotides designed toincrease the biological stability of the molecules or to increase thephysical stability of the duplex formed between the antisense and sensenucleic acids, e.g., phosphorothioate derivatives and acridinesubstituted nucleotides can be used. The antisense nucleic acid also canbe produced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest, described further inthe following subsection).

The antisense nucleic acid molecules of the invention are typicallyadministered to a subject (e.g., by direct injection at a tissue site),or generated in situ such that they hybridize with or bind to cellularmRNA and/or genomic DNA encoding a 52906, 33408, or 12189 protein tothereby inhibit expression of the protein, e.g., by inhibitingtranscription and/or translation. Alternatively, antisense nucleic acidmolecules can be modified to target selected cells and then administeredsystemically. For systemic administration, antisense molecules can bemodified such that they specifically bind to receptors or antigensexpressed on a selected cell surface, e.g., by linking the antisensenucleic acid molecules to peptides or antibodies which bind to cellsurface receptors or antigens. The antisense nucleic acid molecules canalso be delivered to cells using the vectors described herein. Toachieve sufficient intracellular concentrations of the antisensemolecules, vector constructs in which the antisense nucleic acidmolecule is placed under the control of a strong pol II or pol IIIpromoter are preferred.

In yet another embodiment, the antisense nucleic acid molecule of theinvention is an α-anomeric nucleic acid molecule. An α-anomeric nucleicacid molecule forms specific double-stranded hybrids with complementaryRNA in which, contrary to the usual β-units, the strands run parallel toeach other (Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641).The antisense nucleic acid molecule can also comprise a2′-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res.15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBSLett. 215:327-330).

In still another embodiment, an antisense nucleic acid of the inventionis a ribozyme. A ribozyme having specificity for a 52906, 33408, or12189-encoding nucleic acid can include one or more sequencescomplementary to the nucleotide sequence of a 52906, 33408, or 12189cDNA disclosed herein (i.e., SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQID NO:6, or SEQ ID NO:7), and a sequence having known catalytic sequenceresponsible for mRNA cleavage (see U.S. Pat. No. 5,093,246 or Haselhoffand Gerlach (1988) Nature 334:585-591). For example, a derivative of aTetrahymena L-19 IVS RNA can be constructed in which the nucleotidesequence of the active site is complementary to the nucleotide sequenceto be cleaved in a 52906, 33408, or 12189-encoding mRNA. See, e.g., Cechet al. U.S. Pat. No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742.Alternatively, 52906, 33408, or 12189 mRNA can be used to select acatalytic RNA having a specific ribonuclease activity from a pool of RNAmolecules. See, e.g., Bartel, D. and Szostak, J. W. (1993) Science261:1411-1418.

52906, 33408, or 12189 gene expression can be inhibited by targetingnucleotide sequences complementary to the regulatory region of the52906, 33408, or 12189 (e.g., the 52906, 33408, or 12189 promoter and/orenhancers) to form triple helical structures that prevent transcriptionof the 52906, 33408, or 12189 gene in target cells. See generally,Helene, C. (1991) Anticancer Drug Des. 6:569-84; Helene, C. i (1992)Ann. N.Y. Acad. Sci. 660:27-36; and Maher, L. J. (1992) Bioassays14:807-15. The potential sequences that can be targeted for triple helixformation can be increased by creating a so-called “switchback” nucleicacid molecule. Switchback molecules are synthesized in an alternating5′-3′, 3′-5′ manner, such that they base pair with first one strand of aduplex and then the other, eliminating the necessity for a sizeablestretch of either purines or pyrimidines to be present on one strand ofa duplex.

The invention also provides detectably labeled oligonucleotide primerand probe molecules. Typically, such labels are chemiluminescent,fluorescent, radioactive, or colorimetric.

A 52906, 33408, or 12189 nucleic acid molecule can be modified at thebase moiety, sugar moiety or phosphate backbone to improve, e.g., thestability, hybridization, or solubility of the molecule. Fornon-limiting examples of synthetic oligonucleotides with modificationssee Toulmé (2001) Nature Biotech. 19:17 and Faria et al. (2001) NatureBiotech. 19:40-44. Such phosphoramidite oligonucleotides can beeffective antisense agents.

For example, the deoxyribose phosphate backbone of the nucleic acidmolecules can be modified to generate peptide nucleic acids (see HyrupB. et al. (1996) Bioorganic & Medicinal Chemistry 4: 5-23). As usedherein, the terms “peptide nucleic acid” or “PNA” refers to a nucleicacid mimic, e.g., a DNA mimic, in which the deoxyribose phosphatebackbone is replaced by a pseudopeptide backbone and only the fournatural nucleobases are retained. The neutral backbone of a PNA canallow for specific hybridization to DNA and RNA under conditions of lowionic strength. The synthesis of PNA oligomers can be performed usingstandard solid phase peptide synthesis protocols as described in HyrupB. et al. (1996) supra and Perry-O'Keefe et al. Proc. Natl. Acad. Sci.93: 14670-675.

PNAs of 52906, 33408, or 12189 nucleic acid molecules can be used intherapeutic and diagnostic applications. For example, PNAs can be usedas antisense or antigene agents for sequence-specific modulation of geneexpression by, for example, inducing transcription or translation arrestor inhibiting replication. PNAs of 52906, 33408, or 12189 nucleic acidmolecules can also be used in the analysis of single base pair mutationsin a gene, (e.g., by PNA-directed PCR clamping); as ‘artificialrestriction enzymes' when used in combination with other enzymes, (e.g.,S1 nucleases (Hyrup B. et al. (1996) supra)); or as probes or primersfor DNA sequencing or hybridization (Hyrup B. et al. (1996) supra;Perry-O'Keefe supra).

In other embodiments, the oligonucleotide may include other appendedgroups such as peptides (e.g., for targeting host cell receptors invivo), or agents facilitating transport across the cell membrane (see,e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA 86:6553-6556;Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA 84:648-652; PCTPublication No. W088/09810) or the blood-brain barrier (see, e.g., PCTPublication No. W089/10134). In addition, oligonucleotides can bemodified with hybridization-triggered cleavage agents (see, e.g., Krolet al. (1988) Bio-Techniques 6:958-976) or intercalating agents. (see,e.g., Zon (1988) Pharm. Res. 5:539-549). To this end, theoligonucleotide may be conjugated to another molecule, (e.g., a peptide,hybridization triggered cross-linking agent, transport agent, orhybridization-triggered cleavage agent).

The invention also includes molecular beacon oligonucleotide primer andprobe molecules having at least one region which is complementary to a52906, 33408, or 12189 nucleic acid of the invention, two complementaryregions one having a fluorophore and one a quencher such that themolecular beacon is useful for quantitating the presence of the 52906,33408, or 12189 nucleic acid of the invention in a sample. Molecularbeacon nucleic acids are described, for example, in Lizardi et al., U.S.Pat. No. 5,854,033; Nazarenko et al., U.S. Pat. No. 5,866,336, and Livaket al., U.S. Pat. No. 5,876,930.

Isolated 52906, 33408, or 12189 Polypeptides

In another aspect, the invention features, an isolated 52906, 33408, or12189 protein, or fragment, e.g., a biologically active portion, for useas immunogens or antigens to raise or test (or more generally to bind)anti-52906, 33408, or 12189 antibodies. 52906, 33408, or 12189 proteincan be isolated from cells or tissue sources using standard proteinpurification techniques. 52906, 33408, or 12189 protein or fragmentsthereof can be produced by recombinant DNA techniques or synthesizedchemically.

Polypeptides of the invention include those which arise as a result ofthe existence of multiple genes, alternative transcription events,alternative RNA splicing events, and alternative translational andpost-translational events. The polypeptide can be expressed in systems,e.g., cultured cells, which result in substantially the samepost-translational modifications present when expressed the polypeptideis expressed in a native cell, or in systems which result in thealteration or omission of post-translational modifications, e.g.,glycosylation or cleavage, present when expressed in a native cell.

In a preferred embodiment, a 52906, 33408, or 12189 polypeptide has oneor more of the following characteristics:

(i) it has the ability to modulate the flow of K⁺ ions through a cellmembrane, e.g., to allow for the flow of K⁺ ions in and/or out of a cellunder certain conditions;

(ii) it has the ability to modulate the transmission of signals in anelectrically excitable cell, e.g., a neuronal cell or a muscle cell;

(iii) it has a molecular weight, e.g., a deduced molecular weight,preferably ignoring any contribution of post translationalmodifications, amino acid composition or other physical characteristicof a 52906, 33408, or 12189 polypeptide, e.g., a polypeptide of SEQ IDNO:2, SEQ ID NO:5, or SEQ ID NO:8;

(iv) it has an overall sequence similarity of at least 60%, morepreferably at least 70, 80, 90, or 95%, with a polypeptide a of SEQ IDNO:2, SEQ ID NO:5, or SEQ ID NO:8;

(v) it can be found in neuronal cells or muscle cells (e.g., heartcells);

(vi) it has the ability to modulate the resting potential of membranes;

(vii) it has a P-loop domain which is preferably about 70%, 80%, 90% or95% similar with amino acids 616-639 of SEQ ID NO:2, amino acids 420-440of SEQ ID NO:5, or amino acids 339-355 of SEQ ID NO:8;

(viii) it has an ion transport protein domain which is preferably about70%, 80%, 90% or 95% similar with amino acids 472-661 of SEQ ID NO:2,amino acids 247-467 of SEQ ID NO:5, or amino acids 198-383 of SEQ IDNO:8;

(ix) it has a cyclic nucleotide-binding domain which is preferably about70%, 80%, 90% or 95% similar with amino acids 565-655 of SEQ ID NO:5;

(x) it has a potassium channel tetramerisation domain which ispreferably about 70%, 80%, 90% or 95% similar with amino acids 3-101 ofSEQ ID NO:8; or

(xi) it has least 70%, preferably 80%, and most preferably 90% of thecysteines found amino acid sequence of the native protein.

In a preferred embodiment the 52906, 33408, or 12189 protein, orfragment thereof, differs from the corresponding sequence in SEQ IDNO:2, SEQ ID NO:5, or SEQ ID NO:8. In one embodiment it differs by atleast one but by less than 15, 10 or 5 amino acid residues. In anotherit differs from the corresponding sequence in SEQ ID NO:2, SEQ ID NO:5,or SEQ ID NO:8 by at least one residue but less than 20%, 15%, 10% or 5%of the residues in it differ from the corresponding sequence in SEQ IDNO:2, SEQ ID NO:5, or SEQ ID NO:8. (If this comparison requiresalignment the sequences should be aligned for maximum homology. “Looped”out sequences from deletions or insertions, or mismatches, areconsidered differences.) The differences are, preferably, differences orchanges at a non essential residue or a conservative substitution. In apreferred embodiment the differences are not in the ion transportprotein domain. In another preferred embodiment one or more differencesare in the ion transport protein domain.

Other embodiments include a protein that contain one or more changes inamino acid sequence, e.g., a change in an amino acid residue which isnot essential for activity. Such 52906, 33408, or 12189 proteins differin amino acid sequence from SEQ ID NO:2, SEQ ID NO:5, or SEQ ID NO:8,yet retain biological activity.

In one embodiment, the protein includes an amino acid sequence at leastabout 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more homologous toSEQ ID NO:2, SEQ ID NO:5, or SEQ ID NO:8.

A 52906 protein or fragment is provided which varies from the sequenceof SEQ ID NO:2 in regions defined by amino acids about 1-471 and/or662-847 by at least one but by less than 15, 10 or 5 amino acid residuesin the protein or fragment but which does not differ from SEQ ID NO:2 inregions defined by amino acids about 472-661. A 33408 protein orfragment is provided which varies from the sequence of SEQ ID NO:5 inregions defined by amino acids about 1-246 and/or 468-988 by at leastone but by less than 15, 10 or 5 amino acid residues in the protein orfragment but which does not differ from SEQ ID NO:2 in regions definedby amino acids about 247-467. A 12189 protein or fragment is providedwhich varies from the sequence of SEQ ID NO:8 in regions defined byamino acids about 1-197 and/or 384-446 by at least one but by less than15, 10 or 5 amino acid residues in the protein or fragment but whichdoes not differ from SEQ ID NO:2 in regions defined by amino acids about198-383. (If this comparison requires alignment the sequences should bealigned for maximum homology. “Looped” out sequences from deletions orinsertions, or mismatches, are considered differences.) In someembodiments the difference is at a non-essential residue or is aconservative substitution, while in others the difference is at anessential residue or is a non-conservative substitution.

In one embodiment, a biologically active portion of a 52906, 33408, or12189 protein includes an ion transport protein domain, a cyclicnucleotide-binding domain, a potassium channel tetramerisation domain, atransmembrane domain, a cytoplasmic domain, an extracellular domain, aPore-loop domain, or a PAS domain. Moreover, other biologically activeportions, in which other regions of the protein are deleted, can beprepared by recombinant techniques and evaluated for one or more of thefunctional activities of a native 52906, 33408, or 12189 protein.

In a preferred embodiment, the 52906, 33408, or 12189 protein has anamino acid sequence shown in SEQ ID NO:2, SEQ ID NO:5, or SEQ ID NO:8.In other embodiments, the 52906, 33408, or 12189 protein issubstantially identical to SEQ ID NO:2, SEQ ID NO:5, or SEQ ID NO:8. Inyet another embodiment, the 52906, 33408, or 12189 protein issubstantially identical to SEQ ID NO:2, SEQ ID NO:5, or SEQ ID NO:8 andretains the functional activity of the protein of SEQ ID NO:2, SEQ IDNO:5, or SEQ ID NO:8, as described in detail in the subsections above.

52906, 33408, or 12189 Chimeric or Fusion Proteins

In another aspect, the invention provides 52906, 33408, or 12189chimeric or fusion proteins. As used herein, a 52906, 33408, or 12189“chimeric protein” or “fusion protein” includes a 52906, 33408, or 12189polypeptide linked to a non-52906, 33408, or 12189 polypeptide. A“non-52906, 33408, or 12189 polypeptide” refers to a polypeptide havingan amino acid sequence corresponding to a protein which is notsubstantially homologous to the 52906, 33408, or 12189 protein, e.g., aprotein which is different from the 52906, 33408, or 12189 protein andwhich is derived from the same or a different organism. The 52906,33408, or 12189 polypeptide of the fusion protein can correspond to allor a portion e.g., a fragment described herein of a 52906, 33408, or12189 amino acid sequence. In a preferred embodiment, a 52906, 33408, or12189 fusion protein includes at least one (or two) biologically activeportion of a 52906, 33408, or 12189 protein. The non-52906, 33408, or12189 polypeptide can be fused to the N-terminus or C-terminus of the52906, 33408, or 12189 polypeptide.

The fusion protein can include a moiety which has a high affinity for aligand. For example, the fusion protein can be a GST-52906, 33408, or12189 fusion protein in which the 52906, 33408, or 12189 sequences arefused to the C-terminus of the GST sequences. Such fusion proteins canfacilitate the purification of recombinant 52906, 33408, or 12189.Alternatively, the fusion protein can be a 52906, 33408, or 12189protein containing a heterologous signal sequence at its N-terminus. Incertain host cells (e.g., mammalian host cells), expression and/orsecretion of 52906, 33408, or 12189 can be increased through use of aheterologous signal sequence.

Fusion proteins can include all or a part of a serum protein, e.g., anIgG constant region, or human serum albumin.

The 52906, 33408, or 12189 fusion proteins of the invention can beincorporated into pharmaceutical compositions and administered to asubject in vivo. The 52906, 33408, or 12189 fusion proteins can be usedto affect the bioavailability of a 52906, 33408, or 12189 substrate.52906, 33408, or 12189 fusion proteins may be useful therapeutically forthe treatment of disorders caused by, for example, (i) aberrantmodification or mutation of a gene encoding a 52906, 33408, or 12189protein; (ii) mis-regulation of the 52906, 33408, or 12189 gene; and(iii) aberrant post-translational modification of a 52906, 33408, or12189 protein.

Moreover, the 52906, 33408, or 12189-fusion proteins of the inventioncan be used as immunogens to produce anti-52906, 33408, or 12189antibodies in a subject, to purify 52906, 33408, or 12189 ligands and inscreening assays to identify molecules which inhibit the interaction of52906, 33408, or 12189 with a 52906, 33408, or 12189 substrate.

Expression vectors are commercially available that already encode afusion moiety (e.g., a GST polypeptide). A 52906, 33408, or12189-encoding nucleic acid can be cloned into such an expression vectorsuch that the fusion moiety is linked in-frame to the 52906, 33408, or12189 protein.

Variants of 52906, 33408, or 12189 Proteins

In another aspect, the invention also features a variant of a 52906,33408, or 12189 polypeptide, e.g., which functions as an agonist(mimetics) or as an antagonist. Variants of the 52906, 33408, or 12189proteins can be generated by mutagenesis, e.g., discrete point mutation,the insertion or deletion of sequences or the truncation of a 52906,33408, or 12189 protein. An agonist of the 52906, 33408, or 12189proteins can retain substantially the same, or a subset, of thebiological activities of the naturally occurring form of a 52906, 33408,or 12189 protein. An antagonist of a 52906, 33408, or 12189 protein caninhibit one or more of the activities of the naturally occurring form ofthe 52906, 33408, or 12189 protein by, for example, competitivelymodulating a 52906, 33408, or 12189-mediated activity of a 52906, 33408,or 12189 protein. Thus, specific biological effects can be elicited bytreatment with a variant of limited function. Preferably, treatment of asubject with a variant having a subset of the biological activities ofthe naturally occurring form of the protein has fewer side effects in asubject relative to treatment with the naturally occurring form of the52906, 33408, or 12189 protein.

Variants of a 52906, 33408, or 12189 protein can be identified byscreening combinatorial libraries of mutants, e.g., truncation mutants,of a 52906, 33408, or 12189 protein for agonist or antagonist activity.

Libraries of fragments e.g., N terminal, C terminal, or internalfragments, of a 52906, 33408, or 12189 protein coding sequence can beused to generate a variegated population of fragments for screening andsubsequent selection of variants of a 52906, 33408, or 12189 protein.Variants in which a cysteine residues is added or deleted or in which aresidue which is glycosylated is added or deleted are particularlypreferred.

Methods for screening gene products of combinatorial libraries made bypoint mutations or truncation, and for screening cDNA libraries for geneproducts having a selected property are known in the art. Such methodsare adaptable for rapid screening of the gene libraries generated bycombinatorial mutagenesis of 52906, 33408, or 12189 proteins. Recursiveensemble mutagenesis (REM), a new technique which enhances the frequencyof functional mutants in the libraries, can be used in combination withthe screening assays to identify 52906, 33408, or 12189 variants (Arkinand Yourvan (1992) Proc. Natl. Acad. Sci. USA 89:7811-7815; Delgrave etal. (1993) Protein Engineering 6:327-331).

Cell based assays can be exploited to analyze a variegated 52906, 33408,or 12189 library. For example, a library of expression vectors can betransfected into a cell line, e.g., a cell line, which ordinarilyresponds to 52906, 33408, or 12189 in a substrate-dependent manner. Thetransfected cells are then contacted with 52906, 33408, or 12189 and theeffect of the expression of the mutant on signaling by the 52906, 33408,or 12189 substrate can be detected, e.g., by measuring potassium channelactivity, e.g., ion flux through a potassium channel. Plasmid DNA canthen be recovered from the cells which score for inhibition, oralternatively, potentiation of signaling by the 52906, 33408, or 12189substrate, and the individual clones further characterized.

In another aspect, the invention features a method of making a 52906,33408, or 12189 polypeptide, e.g., a peptide having a non-wild typeactivity, e.g., an antagonist, agonist, or super agonist of a naturallyoccurring 52906, 33408, or 12189 polypeptide, e.g., a naturallyoccurring 52906, 33408, or 12189 polypeptide. The method includes:altering the sequence of a 52906, 33408, or 12189 polypeptide, e.g.,altering the sequence e.g., by substitution or deletion of one or moreresidues of a non-conserved region, a domain or residue disclosedherein, and testing the altered polypeptide for the desired activity.

In another aspect, the invention features a method of making a fragmentor analog of a 52906, 33408, or 12189 polypeptide a biological activityof a naturally occurring 52906, 33408, or 12189 polypeptide. The methodincludes: altering the sequence, e.g., by substitution or deletion ofone or more residues, of a 52906, 33408, or 12189 polypeptide, e.g.,altering the sequence of a non-conserved region, or a domain or residuedescribed herein, and testing the altered polypeptide for the desiredactivity.

Anti-52906, 33408, or 12189 Antibodies

In another aspect, the invention provides an anti-52906, 33408, or 12189antibody, or a fragment thereof (e.g., an antigen-binding fragmentthereof). The term “antibody” as used herein refers to an immunoglobulinmolecule or immunologically active portion thereof, i.e., anantigen-binding portion. As used herein, the term “antibody” refers to aprotein comprising at least one, and preferably two, heavy (H) chainvariable regions (abbreviated herein as VH), and at least one andpreferably two light (L) chain variable regions (abbreviated herein asVL). The VH and VL regions can be further subdivided into regions ofhypervariability, termed “complementarity determining regions” (“CDR”),interspersed with regions that are more conserved, termed “frameworkregions” (FR). The extent of the framework region and CDR's has beenprecisely defined (see, Kabat, E. A., et al. (1991) Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242, and Chothia, C.er al. (1987) J. Mol. Biol. 196:901-917, which are incorporated hereinby reference). Each VH and VL is composed of three CDR's and four FRs,arranged from amino-terminus to carboxy-terminus in the following order:FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

The anti-52906, 33408, or 12189 antibody can further include a heavy andlight chain constant region, to thereby form a heavy and lightimmunoglobulin chain, respectively. In one embodiment, the antibody is atetramer of two heavy immunoglobulin chains and two light immunoglobulinchains, wherein the heavy and light immunoglobulin chains areinter-connected by, e.g., disulfide bonds. The heavy chain constantregion is comprised of three domains, CH1, CH2 and CH3. The light chainconstant region is comprised of one domain, CL. The variable region ofthe heavy and light chains contains a binding domain that interacts withan antigen. The constant regions of the antibodies typically mediate thebinding of the antibody to host tissues or factors, including variouscells of the immune system (e.g., effector cells) and the firstcomponent (Clq) of the classical complement system.

As used herein, the term “immunoglobulin” refers to a protein consistingof one or more polypeptides substantially encoded by immunoglobulingenes. The recognized human immunoglobulin genes include the kappa,lambda, alpha (IgA1 and IgA2), gamma (IgG1, IgG2, IgG3, IgG4), delta,epsilon and mu constant region genes, as well as the myriadimmunoglobulin variable region genes. Full-length immunoglobulin “lightchains” (about 25 KDa or 214 amino acids) are encoded by a variableregion gene at the NH2-terminus (about 110 amino acids) and a kappa orlambda constant region gene at the COOH--terminus. Full-lengthimmunoglobulin “heavy chains” (about 50 KDa or 446 amino acids), aresimilarly encoded by a variable region gene (about 116 amino acids) andone of the other aforementioned constant region genes, e.g., gamma(encoding about 330 amino acids).

The term “antigen-binding fragment” of an antibody (or simply “antibodyportion,” or “fragment”), as used herein, refers to one or morefragments of a full-length antibody that retain the ability tospecifically bind to the antigen, e.g., 52906, 33408, or 12189polypeptide or fragment thereof. Examples of antigen-binding fragmentsof the anti-52906, 33408, or 12189 antibody include, but are not limitedto: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH,CL and CH1 domains; (ii) a F(ab′)₂ fragment, a bivalent fragmentcomprising two Fab fragments linked by a disulfide bridge at the hingeregion; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) aFv fragment consisting of the VL and VH domains of a single arm of anantibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546),which consists of a VH domain; and (vi) an isolated complementaritydetermining region (CDR). Furthermore, although the two domains of theFv fragment, VL and VH, are coded for by separate genes, they can bejoined, using recombinant methods, by a synthetic linker that enablesthem to be made as a single protein chain in which the VL and VH regionspair to form monovalent molecules (known as single chain Fv (scFv); seee.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988)Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodiesare also encompassed within the term “antigen-binding fragment” of anantibody. These antibody fragments are obtained using conventionaltechniques known to those with skill in the art, and the fragments arescreened for utility in the same manner as are intact antibodies.

The anti-52906, 33408, or 12189 antibody can be a polyclonal or amonoclonal antibody. In other embodiments, the antibody can berecombinantly produced, e.g., produced by phage display or bycombinatorial methods.

Phage display and combinatorial methods for generating anti-52906,33408, or 12189 antibodies are known in the art (as described in, e.g.,Ladner et al. U.S. Pat. No. 5,223,409; Kang et al. InternationalPublication No. WO 92/18619; Dower et al. International Publication No.WO 91/17271; Winter et al. International Publication WO 92/20791;Markland et al. International Publication No. WO 92/15679; Breitling etal. International Publication WO 93/01288; McCafferty et al.International Publication No. WO 92/01047; Garrard et al. InternationalPublication No. WO 92/09690; Ladner et al. International Publication No.WO 90/02809; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al.(1992) Hum Antibod Hybridomas 3:81-85; Huse et al. (1989) Science246:1275-1281; Griffths et al. (1993) EMBO J 12:725-734; Hawkins et al.(1992) J Mol Biol 226:889-896; Clackson et al. (1991) Nature352:624-628; Gram et al. (1992) PNAS 89:3576-3580; Garrad et al. (1991)Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res19:4133-4137; and Barbas et al. (1991) PNAS 88:7978-7982, the contentsof all of which are incorporated by reference herein).

In one embodiment, the anti-52906, 33408, or 12189 antibody is a fullyhuman antibody (e.g., an antibody made in a mouse which has beengenetically engineered to produce an antibody from a humanimmunoglobulin sequence), or a non-human antibody, e.g., a rodent (mouseor rat), goat, primate (e.g., monkey), camel antibody. Preferably, thenon-human antibody is a rodent (mouse or rat antibody). Method ofproducing rodent antibodies are known in the art.

Human monoclonal antibodies can be generated using transgenic micecarrying the human immunoglobulin genes rather than the mouse system.Splenocytes from these transgenic mice immunized with the antigen ofinterest are used to produce hybridomas that secrete human mAbs withspecific affinities for epitopes from a human protein (see, e.g., Woodet al. International Application WO 91/00906, Kucherlapati et at. PCTpublication WO 91/10741; Lonberg et al. International Application WO92/03918; Kay et al. International Application 92/03917; Lonberg, N. etal. 1994 Nature 368:856-859; Green, L. L. et al. 1994 Nature Genet.7:13-21; Morrison, S. L. et al. 1994 Proc. Natl. Acad. Sci. USA81:6851-6855; Bruggeman et al. 1993 Year Immunol 7:33-40; Tuaillon etal. 1993 PNAS90:3720-3724; Bruggeman et al. 1991 Eur J Immunol21:1323-1326).

An anti-52906, 33408, or 12189 antibody can be one in which the variableregion, or a portion thereof, e.g., the CDR's, are generated in anon-human organism, e.g., a rat or mouse. Chimeric, CDR-grafted, andhumanized antibodies are within the invention. Antibodies generated in anon-human organism, e.g., a rat or mouse, and then modified, e.g., inthe variable framework or constant region, to decrease antigenicity in ahuman are within the invention.

Chimeric antibodies can be produced by recombinant DNA techniques knownin the art. For example, a gene encoding the Fc constant region of amurine (or other species) monoclonal antibody molecule is digested withrestriction enzymes to remove the region encoding the murine Fc, and theequivalent portion of a gene encoding a human Fc constant region issubstituted (see Robinson et al., International Patent PublicationPCT/US86/02269; Akira, et al., European Patent Application 184,187;Taniguchi, M., European Patent Application 171,496; Morrison et al.,European Patent Application 173,494; Neuberger et al., InternationalApplication WO 86/01533; Cabilly et al. U.S. Pat. No. 4,816,567; Cabillyet al., European Patent Application 125,023; Better et al. (1988 Science240:1041-1043); Liu et al. (1987) PNAS 84:3439-3443; Liu et al., 1987,J. Immunol. 139:3521-3526; Sun et al. (1987) PNAS 84:214-218; Nishimuraet al., 1987, Canc. Res. 47:999-1005; Wood et al. (1985) Nature314:446-449; and Shaw et al., 1988, J. Natl Cancer Inst. 80:1553-1559).

A humanized or CDR-grafted antibody will have at least one or two butgenerally all three recipient CDR's (of heavy and or lightimmunoglobulin chains) replaced with a donor CDR. The antibody may bereplaced with at least a portion of a non-human CDR or only some of theCDR's may be replaced with non-human CDR's. It is only necessary toreplace the number of CDR's required for binding of the humanizedantibody to a 52906, 33408, or 12189 or a fragment thereof. Preferably,the donor will be a rodent antibody, e.g., a rat or mouse antibody, andthe recipient will be a human framework or a human consensus framework.Typically, the immunoglobulin providing the CDR's is called the “donor”and the immunoglobulin providing the framework is called the “acceptor.”In one embodiment, the donor immunoglobulin is a non-human (e.g.,rodent). The acceptor framework is a naturally-occurring (e.g., a human)framework or a consensus framework, or a sequence about 85% or higher,preferably 90%, 95%, 99% or higher identical thereto.

As used herein, the term “consensus sequence” refers to the sequenceformed from the most frequently occurring amino acids (or nucleotides)in a family of related sequences (See e.g., Winnaker, From Genes toClones (Verlagsgesellschaft, Weinheim, Germany 1987). In a family ofproteins, each position in the consensus sequence is occupied by the.amino acid occurring most frequently at that position in the family. Iftwo amino acids occur equally frequently, either can be included in theconsensus sequence. A “consensus framework” refers to the frameworkregion in the consensus immunoglobulin sequence.

An antibody can be humanized by methods known in the art. Humanizedantibodies can be generated by replacing sequences of the Fv variableregion which are not directly involved in antigen binding withequivalent sequences from human Fv variable regions. General methods forgenerating humanized antibodies are provided by Morrison, S. L., 1985,Science 229:1202-1207, by Oi et al., 1986, BioTechniques 4:214, and byQueen et al. U.S. Pat. No. 5,585,089, U.S. Pat. No. 5,693,761 and U.S.Pat. No. 5,693,762, the contents of all of which are hereby incorporatedby reference. Those methods include isolating, manipulating, andexpressing the nucleic acid sequences that encode all or part ofimmunoglobulin Fv variable regions from at least one of a heavy or lightchain. Sources of such nucleic acid are well known to those skilled inthe art and, for example, may be obtained from a hybridoma producing anantibody against a 52906, 33408, or 12189 polypeptide or fragmentthereof. The recombinant DNA encoding the humanized antibody, orfragment thereof, can then be cloned into an appropriate expressionvector. Humanized or CDR-grafted antibodies can be produced byCDR-grafting or CDR substitution, wherein one, two, or all CDR's of animmunoglobulin chain can be replaced. See e.g., U.S. Pat. No. 5,225,539;Jones et al. 1986 Nature 321:552-525; Verhoeyan et al. 1988 Science239:1534; Beidler et al. 1988 J. Immunol. 141:4053-4060; Winter U.S.Pat. No. 5,225,539, the contents of all of which are hereby expresslyincorporated by reference. Winter describes a CDR-grafting method whichmay be used to prepare the humanized antibodies of the present invention(UK Patent Application GB 2188638A, filed on Mar. 26, 1987; Winter U.S.Pat. No. 5,225,539), the contents of which is expressly incorporated byreference.

Also within the scope of the invention are humanized antibodies in whichspecific amino acids have been substituted, deleted or added. Preferredhumanized antibodies have amino acid substitutions in the frameworkregion, such as to improve binding to the antigen. For example, ahumanized antibody will have framework residues identical to the donorframework residue or to another amino acid other than the recipientframework residue. To generate such antibodies, a selected, small numberof acceptor framework residues of the humanized immunoglobulin chain canbe replaced by the corresponding donor amino acids. Preferred locationsof the substitutions include amino acid residues adjacent to the CDR, orwhich are capable of interacting with a CDR (see e.g., U.S. Pat. No.5,585,089). Criteria for selecting amino acids from the donor aredescribed in U.S. Pat. No. 5,585,089, e.g., columns 12-16 of U.S. Pat.No. 5,585,089, the e.g., columns 12-16 of U.S. Pat. No. 5,585,089, thecontents of which are hereby incorporated by reference. Other techniquesfor humanizing antibodies are described in Padlan et al. EP 519596 A1,published on Dec. 23, 1992.

In preferred embodiments an antibody can be made by immunizing withpurified 52906, 33408, or 12189 antigen, or a fragment thereof, e.g., afragment described herein, membrane associated antigen, tissue, e.g.,crude tissue preparations, whole cells, preferably living cells, lysedcells, or cell fractions, e.g., membrane fractions.

A full-length 52906, 33408, or 12189 protein or, antigenic peptidefragment of 52906, 33408, or 12189 can be used as an immunogen or can beused to identify anti-52906, 33408, or 12189 antibodies made with otherimmunogens, e.g., cells, membrane preparations, and the like. Theantigenic peptide of 52906, 33408, or 12189 should include at least 8amino acid residues of the amino acid sequence shown in SEQ ID NO:2, SEQID NO:5, or SEQ ID NO:8 and encompasses an epitope of 52906, 33408, or12189. Preferably, the antigenic peptide includes at least 10 amino acidresidues, more preferably at least 15 amino acid residues, even morepreferably at least 20 amino acid residues, and most preferably at least30 amino acid residues.

Fragments of 52906, 33408, or 12189 which include residues about 241-265of SEQ ID NO:2, 710-740 of SEQ ID NO:5, or 35-55 of SEQ ID NO:8 can beused to make, e.g., used as immunogens or used to characterize thespecificity of an antibody, antibodies against hydrophilic regions ofthe 52906, 33408, or 12189 protein. Similarly, fragments of 52906,33408, or 12189 which include residues about 785-800 of SEQ ID NO:2,585-600 of SEQ ID NO:5, or 75-95 of SEQ ID NO:8 can be used to make anantibody against a hydrophobic region of the 52906, 33408, or 12189protein. Fragments of 52906, 33408, or 12189 which include residues420-432 of SEQ ID NO:2, 237-244 of SEQ ID NO:5, or 153-199 of SEQ IDNO:8 can be used to make an antibody against an extracellular region ofthe 52906, 33408, or 12189 protein. Fragments of 52906, 33408, or 12189which include residues 1-401 of SEQ ID NO:2, 1-218 of SEQ ID NO:5, or1-133 of SEQ ID NO:8 can be used to make an antibody against anintracellular region of the 52906, 33408, or 12189 protein. Fragments of52906, 33408, or 12189 which include residues 616-639 of SEQ ID NO:2,420-440 of SEQ ID NO:5, or 339-355 of SEQ ID NO:8 can be used to make anantibody against the P-loop region of the 52906, 33408, or 12189protein. Fragments of 52906, 33408, or 12189 which include amino acids472-661 of SEQ ID NO:2, amino acids 247-467 of SEQ ID NO:5, or aminoacids 198-383 of SEQ ID NO:8 can be used to make an antibody against theion transport protein domain of the 52906, 33408, or 12189 protein.Fragments of 33408 which include amino acids 565-655 of SEQ ID NO:5 canbe used to make an antibody against the cyclic nucleotide-binding domainof the 33408 protein. Fragments of 12189 which include amino acids 3-101of SEQ ID NO:8 can be used to make an antibody against the potassiumchannel tetramerisation domain of the 12189 protein.

Antibodies reactive with, or specific for, any of these regions, orother regions or domains described herein are provided.

Antibodies which bind only native 52906, 33408, or 12189 protein, onlydenatured or otherwise non-native 52906, 33408, or 12189 protein, orwhich bind both, are with in the invention. Antibodies with linear orconformational epitopes are within the invention. Conformationalepitopes can sometimes be identified by identifying antibodies whichbind to native but not denatured 52906, 33408, or 12189 protein.

Preferred epitopes encompassed by the antigenic peptide are regions of52906, 33408, or 12189 are located on the surface of the protein, e.g.,hydrophilic regions, as well as regions with high antigenicity. Forexample, an Emini surface probability analysis of the human 52906,33408, or 12189 protein sequence can be used to indicate theregions-that have a particularly high probability of being localized tothe surface of the 52906, 33408, or 12189 protein and are thus likely toconstitute surface residues useful for targeting antibody production.

In a preferred embodiment the antibody can bind to the extracellularportion of the 52906, 33408, or 12189 protein, e.g., it can bind to awhole cell which expresses the 52906, 33408, or 12189 protein. Inanother embodiment, the antibody binds an intracellular portion of the52906, 33408, or 12189 protein.

In preferred embodiments antibodies can bind one or more of purifiedantigen, membrane associated antigen, tissue, e.g., tissue sections,whole cells, preferably living cells, lysed cells, cell fractions, e.g.,membrane fractions.

The anti-52906, 33408, or 12189 antibody can be a single chain antibody.A single-chain antibody (scFV) may be engineered (see, for example,Colcher, D. et al. (1999) Ann N Y Acad Sci 880:263-80; and Reiter, Y.(1996) Clin Cancer Res 2:245-52). The single chain antibody can bedimerized or multimerized to generate multivalent antibodies havingspecificities for different epitopes of the same target 52906, 33408, or12189 protein.

In a preferred embodiment the antibody has: effector function; and canfix complement. In other embodiments the antibody does not; recruiteffector cells; or fix complement.

In a preferred embodiment, the antibody has reduced or no ability tobind an Fc receptor. For example., it is a isotype or subtype, fragmentor other mutant, which does not support binding to an Fc receptor, e.g.,it has a mutagenized or deleted Fc receptor binding region.

In a preferred embodiment, an anti-52906, 33408, or 12189 antibodyalters (e.g., increases or decreases) an activity of a 52906, 33408, or12189 polypeptide, e.g., the ability to modulate the flow of K⁺ ionsthrough a cell membrane and/or the ability to modulate the transmissionof signals in an electrically excitable cell, e.g., a neuronal cell or amuscle cell. For example, the antibody can bind at or in proximity to aPore loop domain, e.g., to an epitope that includes a residue locatedfrom about 616-639 of SEQ ID NO:2, 420-440 of SEQ ID NO:5, or 339-355 ofSEQ ID NO:8.

The antibody can be coupled to a toxin, e.g., a polypeptide toxin, e,g,ricin or diphtheria toxin or active fragment hereof, or a radioactivenucleus, or imaging agent, e.g. a radioactive, enzymatic, or other,e.g., imaging agent, e.g., a NMR contrast agent. Labels which producedetectable radioactive emissions or fluorescence are preferred.

An anti-52906, 33408, or 12189 antibody (e.g., monoclonal antibody) canbe used to isolate 52906, 33408, or 12189 by standard techniques, suchas affinity chromatography or immunoprecipitation. Moreover, ananti-52906, 33408, or 12189 antibody can be used to detect 52906, 33408,or 12189 protein (e.g., in a cellular lysate or cell supernatant) inorder to evaluate the abundance and pattern of expression of theprotein. Anti-52906, 33408, or 12189 antibodies can be useddiagnostically to monitor protein levels in tissue as part of a clinicaltesting procedure, e.g., to determine the efficacy of a given treatmentregimen. Detection can be facilitated by coupling (i.e., physicallylinking) the antibody to a detectable substance (i.e., antibodylabeling). Examples of detectable substances include various enzymes,prosthetic groups, fluorescent materials, luminescent materials,bioluminescent materials, and radioactive materials. Examples ofsuitable enzymes include horseradish peroxidase, alkaline phosphatase,β-galactosidase, or acetylcholinesterase; examples of suitableprosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin,and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or³H.

The invention also includes a nucleic acids which encodes an anti-52906,33408, or 12189 antibody, e.g., an anti-52906, 33408, or 12189 antibodydescribed herein. Also included are vectors which include the nucleicacid and sells transformed with the nucleic acid, particularly cellswhich are useful for producing an antibody, e.g., mammalian cells, e.g.CHO or lymphatic cells.

The invention also includes cell lines, e.g., hybridomas, which make ananti-52906, 33408, or 12189 antibody, e.g., and antibody describedherein, and method of using said cells to make a 52906, 33408, or 12189antibody.

Recombinant Expression Vectors, Host Cells and Genetically EngineeredCells

In another aspect, the invention includes, vectors, preferablyexpression vectors, containing a nucleic acid encoding a polypeptidedescribed herein. As used herein, the term “vector” refers to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked and can include a plasmid, cosmid or viral vector. Thevector can be capable of autonomous replication or it can integrate intoa host DNA. Viral vectors include, e.g., replication defectiveretroviruses, adenoviruses and adeno-associated viruses.

A vector can include a 52906, 33408, or 12189 nucleic acid in a formsuitable for expression of the nucleic acid in a host cell. Preferablythe recombinant expression vector includes one or more regulatorysequences operatively linked to the nucleic acid sequence to beexpressed. The term “regulatory sequence” includes promoters, enhancersand other expression control elements (e.g., polyadenylation signals).Regulatory sequences include those which direct constitutive expressionof a nucleotide sequence, as well as tissue-specific regulatory and/orinducible sequences. The design of the expression vector can depend onsuch factors as the choice of the host cell to be transformed, the levelof expression of protein desired, and the like. The expression vectorsof the invention can be introduced into host cells to thereby produceproteins or polypeptides, including fusion proteins or polypeptides,encoded by nucleic acids as described herein (e.g., 52906, 33408, or12189 proteins, mutant forms of 52906, 33408, or 12189 proteins, fusionproteins, and the like).

The recombinant expression vectors of the invention can be designed forexpression of 52906, 33408, or 12189 proteins in prokaryotic oreukaryotic cells. For example, polypeptides of the invention can beexpressed in E. coli, insect cells (e.g., using baculovirus expressionvectors), yeast cells or mammalian cells. Suitable host cells arediscussed further in Goeddel, (1990) Gene Expression Technology: Methodsin Enzymology 185, Academic Press, San Diego, Calif. Alternatively, therecombinant expression vector can be transcribed and translated invitro, for example using T7 promoter regulatory sequences and T7polymerase.

Expression of proteins in prokaryotes is most often carried out in E.coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors typically serve three purposes: 1) to increase expression ofrecombinant protein; 2) to increase the solubility of the recombinantprotein; and 3) to aid in the purification of the recombinant protein byacting as a ligand in affinity purification. Often, a proteolyticcleavage site is introduced at the junction of the fusion moiety and therecombinant protein to enable separation of the recombinant protein fromthe fusion moiety subsequent to purification of the fusion protein. Suchenzymes, and their cognate recognition sequences, include Factor Xa,thrombin and enterokinase. Typical fusion expression vectors includepGEX (Pharmacia Biotech Inc; Smith, D. B. and Johnson, K. S. (1988) Gene67:31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5(Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase(GST), maltose E binding protein, or protein A, respectively, to thetarget recombinant protein.

Purified fusion proteins can be used in 52906, 33408, or 12189 activityassays, (e.g., direct assays or competitive assays described in detailbelow), or to generate antibodies specific for 52906, 33408, or 12189proteins. In a preferred embodiment, a fusion protein expressed in aretroviral expression vector of the present invention can be used toinfect bone marrow cells which are subsequently transplanted intoirradiated recipients. The pathology of the subject recipient is thenexamined after sufficient time has passed (e.g., six weeks).

To maximize recombinant protein expression in E. coli is to express theprotein in a host bacteria with an impaired capacity to proteolyticallycleave the recombinant protein (Gottesman, S., (1990) Gene ExpressionTechnology: Methods in Enzymology 185, Academic Press, San Diego, Calif.119-128). Another strategy is to alter the nucleic acid sequence of thenucleic acid to be inserted into an expression vector so that theindividual codons for each amino acid are those preferentially utilizedin E. coli (Wada et al., (1992) Nucleic Acids Res. 20:2111-2118). Suchalteration of nucleic acid sequences of the invention can be carried outby standard DNA synthesis techniques.

The 52906, 33408, or 12189 expression vector can be a yeast expressionvector, a vector for expression in insect cells, e.g., a baculovirusexpression vector or a vector suitable for expression in mammaliancells.

When used in mammalian cells, the expression vector's control functionscan be provided by viral regulatory elements. For example, commonly usedpromoters are derived from polyoma, Adenovirus 2, cytomegalovirus andSimian Virus 40.

In another embodiment, the promoter is an inducible promoter, e.g., apromoter regulated by a steroid hormone, by a polypeptide hormone (e.g.,by means of a signal transduction pathway), or by a heterologouspolypeptide (e.g., the tetracycline-inducible systems, “Tet-On” and“Tet-Off”; see, e.g., Clontech Inc., CA, Gossen and Bujard (1992) Proc.Natl. Acad. Sci. USA 89:5547, and Paillard (1989) Human Gene Therapy9:983).

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Non-limiting examples of suitabletissue-specific promoters include the albumin promoter (liver-specific;Pinkert et al. (1987) Genes Dev. 1:268-277), lymphoid-specific promoters(Calame and Eaton (1988) Adv. Immunol. 43:235-275), in particularpromoters of T cell receptors (Winoto and Baltimore (1989) EMBO J.8:729-733) and immunoglobulins (Banerji et al. (1983) Cell 33:729-740;Queen and Baltimore (1983) Cell 33:741-748), neuron-specific promoters(e.g., the neurofilament promoter; Byrne and Ruddle (1989) Proc. Natl.Acad. Sci. USA 86:5473-5477), pancreas-specific promoters (Edlund et al.(1985) Science 230:912-916), and mammary gland-specific promoters (e.g.,milk whey promoter; U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166). Developmentally-regulated promoters are alsoencompassed, for example, the murine hox promoters (Kessel and Gruss(1990) Science 249:374-379) and the α-fetoprotein promoter (Campes andTilghman (1989) Genes Dev. 3:537-546).

The invention further provides a recombinant expression vectorcomprising a DNA molecule of the invention cloned into the expressionvector in an antisense orientation. Regulatory sequences (e.g., viralpromoters and/or enhancers) operatively linked to a nucleic acid clonedin the antisense orientation can be chosen which direct theconstitutive, tissue specific or cell type specific expression ofantisense RNA in a variety of cell types. The antisense expressionvector can be in the form of a recombinant plasmid, phagemid orattenuated virus.

Another aspect the invention provides a host cell which includes anucleic acid molecule described herein, e.g., a 52906, 33408, or 12189nucleic acid molecule within a recombinant expression vector or a 52906,33408, or 12189 nucleic acid molecule containing sequences which allowit to homologously recombine into a specific site of the host cell'sgenome. The terms “host cell” and “recombinant host cell” are usedinterchangeably herein. Such terms refer not only to the particularsubject cell but to the progeny or potential progeny of such a cell.Because certain modifications may occur in succeeding generations due toeither mutation or environmental influences, such progeny may not, infact, be identical to the parent cell, but are still included within thescope of the term as used herein.

A host cell can be any prokaryotic or eukaryotic cell. For example, a52906, 33408, or 12189 protein can be expressed in bacterial cells (suchas E. coli), insect cells, yeast or mammalian cells (such as Chinesehamster ovary cells (CHO) or COS cells (African green monkey kidneycells CV-1 origin SV40 cells; Gluzman (1981) CellI23:175-182)). Othersuitable host cells are known to those skilled in the art.

Vector DNA can be introduced into host cells via conventionaltransformation or transfection techniques. As used herein, the terms“transformation” and “transfection” are intended to refer to a varietyof art-recognized techniques for introducing foreign nucleic acid (e.g.,DNA) into a host cell, including calcium phosphate or calcium chlorideco-precipitation, DEAE-dextran-mediated transfection, lipofection, orelectroporation.

A host cell of the invention can be used to produce (i.e., express) a52906, 33408, or 12189 protein. Accordingly, the invention furtherprovides methods for producing a 52906, 33408, or 12189 protein usingthe host cells of the invention. In one embodiment, the method includesculturing the host cell of the invention (into which a recombinantexpression vector encoding a 52906, 33408, or 12189 protein has beenintroduced) in a suitable medium such that a 52906, 33408, or 12189protein is produced. In another embodiment, the method further includesisolating a 52906, 33408, or 12189 protein from the medium or the hostcell.

In another aspect, the invention features, a cell or purifiedpreparation of cells which include a 52906, 33408, or 12189 transgene,or which otherwise misexpress 52906, 33408, or 12189. The cellpreparation can consist of human or non-human cells, e.g., rodent cells,e.g., mouse or rat cells, rabbit cells, or pig cells. In preferredembodiments, the cell or cells include a 52906, 33408, or 12189transgene, e.g., a heterologous form of a 52906, 33408, or 12189, e.g.,a gene derived from humans (in the case of a non-human cell). The 52906,33408, or 12189 transgene can be misexpressed, e.g., overexpressed orunderexpressed. In other preferred embodiments, the cell or cellsinclude a gene that mis-expresses an endogenous 52906, 33408, or 12189,e.g., a gene the expression of which is disrupted, e.g., a knockout.Such cells can serve as a model for studying disorders that are relatedto mutated or mis-expressed 52906, 33408, or 12189 alleles or for use indrug screening.

In another aspect, the invention features, a human cell, e.g., aneuronal cell or a muscle cell, transformed with nucleic acid whichencodes a subject 52906, 33408, or 12189 polypeptide.

Also provided are cells, preferably human cells, e.g., a neuronal cell,a muscle cell, a hematopoietic cell, or a fibroblast cell, in which anendogenous 52906, 33408, or 12189 is under the control of a regulatorysequence that does not normally control the expression of the endogenous52906, 33408, or 12189 gene. The expression characteristics of anendogenous gene within a cell, e.g., a cell line or microorganism, canbe modified by inserting a heterologous DNA regulatory element into thegenome of the cell such that the inserted regulatory element is operablylinked to the endogenous 52906, 33408, or 12189 gene. For example, anendogenous 52906, 33408, or 12189 gene which is “transcriptionallysilent,” e.g., not normally expressed, or expressed only at very lowlevels, may be activated by inserting a regulatory element which iscapable of promoting the expression of a normally expressed gene productin that cell. Techniques such as targeted homologous recombinations, canbe used to insert the heterologous DNA as described in, e.g., Chappel,U.S. Pat. No. 5,272,071; WO 91/06667, published in May 16, 1991.

In a preferred embodiment, recombinant cells described herein can beused for replacement therapy in a subject. For example, a nucleic acidencoding a 52906, 33408, or 12189 polypeptide operably linked to aninducible promoter (e.g., a steroid hormone receptor-regulated promoter)is introduced into a human or nonhuman, e.g., mammalian, e.g., porcinerecombinant cell. The cell is cultivated and encapsulated in abiocompatible material, such as poly-lysine alginate, and subsequentlyimplanted into the subject. See, e.g., Lanza (1996) Nat. Biotechnol.14:1107; Joki et al. (2001) Nat. Biotechnol. 19:35; and U.S. Pat. No.5,876,742. Production of 52906, 33408, or 12189 polypeptide can beregulated in the subject by administering an agent (e.g., a steroidhormone) to the subject. In another preferred embodiment, the implantedrecombinant cells express and secrete an antibody specific for a 52906,33408, or 12189 polypeptide. The antibody can be any antibody or anyantibody derivative described herein.

Transgenic Animals

The invention provides non-human transgenic animals. Such animals areuseful for studying the function and/or activity of a 52906, 33408, or12189 protein and for identifying and/or evaluating modulators of 52906,33408, or 12189 activity. As used herein, a “transgenic animal” is anon-human animal, preferably a mammal, more preferably a rodent such asa rat or mouse, in which one or more of the cells of the animal includesa transgene. Other examples of transgenic animals include non-humanprimates, sheep, dogs, cows, goats, chickens, amphibians, and the like.A transgene is exogenous DNA or a rearrangement, e.g., a deletion ofendogenous chromosomal DNA, which preferably is integrated into oroccurs in the genome of the cells of a transgenic animal. A transgenecan direct the expression of an encoded gene product in one or more celltypes or tissues of the transgenic animal, other transgenes, e.g., aknockout, reduce expression. Thus, a transgenic animal can be one inwhich an endogenous 52906, 33408, or 12189 gene has been altered by,e.g., by homologous recombination between the endogenous gene and anexogenous DNA molecule introduced into a cell of the animal, e.g., anembryonic cell of the animal, prior to development of the animal.

Intronic sequences and polyadenylation signals can also be included inthe transgene to increase the efficiency of expression of the transgene.A tissue-specific regulatory sequence(s) can be operably linked to atransgene of the invention to direct expression of a 52906, 33408, or12189 protein to particular cells. A transgenic founder animal can beidentified based upon the presence of a 52906, 33408, or 12189 transgenein its genome and/or expression of 52906, 33408, or 12189 mRNA intissues or cells of the animals. A transgenic founder animal can then beused to breed additional animals carrying the transgene. Moreover,transgenic animals carrying a transgene encoding a 52906, 33408, or12189 protein can further be bred to other transgenic animals carryingother transgenes.

52906, 33408, or 12189 proteins or polypeptides can be expressed intransgenic animals or plants, e.g., a nucleic acid encoding the proteinor polypeptide can be introduced into the genome of an animal. Inpreferred embodiments the nucleic acid is placed under the control of atissue specific promoter, e.g., a milk or egg specific promoter, andrecovered from the milk or eggs produced by the animal. Suitable animalsare mice, pigs, cows, goats, and sheep.

The invention also includes a population of cells from a transgenicanimal, as discussed, e.g., below.

Uses

The nucleic acid molecules, proteins, protein homologues, and antibodiesdescribed herein can be used in one or more of the following methods: a)screening assays; b) predictive medicine (e.g., diagnostic assays,prognostic assays, monitoring clinical trials, and pharmacogenetics);and c) methods of treatment (e.g., therapeutic and prophylactic).

The isolated nucleic acid molecules of the invention can be used, forexample, to express a 52906, 33408, or 12189 protein (e.g., via arecombinant expression vector in a host cell in gene therapyapplications), to detect a 52906, 33408, or 12189 mRNA (e.g., in abiological sample) or a genetic alteration in a 52906, 33408, or 12189gene, and to modulate 52906, 33408, or 12189 activity, as describedfurther below. The 52906, 33408, or 12189 proteins can be used to treatdisorders characterized by insufficient or excessive production of a52906, 33408, or 12189 substrate or production of 52906, 33408, or 12189inhibitors. In addition, the 52906, 33408, or 12189 proteins can be usedto screen for naturally occurring 52906, 33408, or 12189 substrates, toscreen for drugs or compounds which modulate 52906, 33408, or 12189activity, as well as to treat disorders characterized by insufficient orexcessive production of 52906, 33408, or 12189 protein or production of52906, 33408, or 12189 protein forms which have decreased, aberrant orunwanted activity compared to 52906, 33408, or 12189 wild type protein(e.g., disorders characterized by abnormal ion flux such as neurologicaldisorders or cardiac disorders). Moreover, the anti-52906, 33408, or12189 antibodies of the invention can be used to detect and isolate52906, 33408, or 12189 proteins, regulate the bioavailability of 52906,33408, or 12189 proteins, and modulate 52906, 33408, or 12189 activity.

A method of evaluating a compound for the ability to interact with,e.g., bind, a subject 52906, 33408, or 12189 polypeptide is provided.The method includes: contacting the compound with the subject 52906,33408, or 12189 polypeptide; and evaluating ability of the compound tointeract with, e.g., to bind or form a complex with the subject 52906,33408, or 12189 polypeptide. This method can be performed in vitro,e.g., in a cell free system, or in vivo, e.g., in a two-hybridinteraction trap assay. This method can be used to identify naturallyoccurring molecules that interact with subject 52906, 33408, or 12189polypeptide. It can also be used to find natural or synthetic inhibitorsof subject 52906, 33408, or 12189 polypeptide. Screening methods arediscussed in more detail below.

Screening Assays

The invention provides methods (also referred to herein as “screeningassays”) for identifying modulators, i.e., candidate or test compoundsor agents (e.g., proteins, peptides, peptidomimetics, peptoids, smallmolecules or other drugs) which bind to 52906, 33408, or 12189 proteins,have a stimulatory or inhibitory effect on, for example, 52906, 33408,or 12189 expression or 52906, 33408, or 12189 activity, or have astimulatory or inhibitory effect on, for example, the expression oractivity of a 52906, 33408, or 12189 substrate. Compounds thusidentified can be used to modulate the activity of target gene products(e.g., 52906, 33408, or 12189 genes) in a therapeutic protocol, toelaborate the biological function of the target gene product, or toidentify compounds that disrupt normal target gene interactions.

In one embodiment, the invention provides assays for screening candidateor test compounds-which are substrates of a 52906, 33408, or 12189protein or polypeptide or a biologically active portion thereof. Inanother embodiment, the invention provides assays for screeningcandidate or test compounds that bind to or modulate an activity of a52906, 33408, or 12189 protein or polypeptide or a biologically activeportion thereof.

In one embodiment, an activity of a 52906, 33408, or 12189 protein canbe assayed by measuring the flow of K⁺ ions through a cell membraneand/or by measuring the transmission of signals in an electricallyexcitable cell, e.g., a neuronal cell or a muscle cell. For example, anactivity of a 52906, 33408, or 12189 protein can be assayed by measuringmembrane currents as described in Köhler et al. (1996) Science273:1709-1714, Saganich et al. (1999) J. Neuroscience 19:10789-10802, orKalman et al.(1998) J. Biol. Chem. 273:5851-5857.

The test compounds of the present invention can be obtained using any ofthe numerous approaches in combinatorial library methods known in theart, including: biological libraries; peptoid libraries (libraries ofmolecules having the functionalities of peptides, but with a novel,non-peptide backbone which are resistant to enzymatic degradation butwhich nevertheless remain bioactive; see, e.g., Zuckermann, R. N. et al.(1994) J. Med. Chem. 37:2678-85); spatially addressable parallel solidphase or solution phase libraries; synthetic library methods requiringdeconvolution; the ‘one-bead one-compound’ library method; and syntheticlibrary methods using affinity chromatography selection. The biologicallibrary and peptoid library approaches are limited to peptide libraries,while the other four approaches are applicable to peptide, non-peptideoligomer or small molecule libraries of compounds (Lam (1997) AnticancerDrug Des. 12:145).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad.Sci. USA. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al.(1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed.Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061;and Gallop et al. (1994) J. Med Chem. 37:1233.

Libraries of compounds may be presented in solution (e.g., Houghten(1992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (Ladner,U.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. 5,223,409),plasmids (Cull et al. (1992) Proc Natl Acad Sci USA 89:1865-1869) or onphage (Scott and Smith (1990) Science 249:386-390; Devlin (1990) Science249:404-406; Cwirla et al. (1990) Proc. Natl. Acad. Sci. 87:6378-6382;Felici (1991) J. Mol. Biol. 222:301-310; Ladner supra.).

In one embodiment, an assay is a cell-based assay in which a cell whichexpresses a 52906, 33408, or 12189 protein or biologically activeportion thereof is contacted with a test compound, and the ability ofthe test compound to modulate 52906, 33408, or 12189 activity isdetermined. Determining the ability of the test compound to modulate52906, 33408, or 12189 activity can be accomplished by monitoring, forexample, potassium channel activity, e.g., ion flux through a potassiumchannel. The cell, for example, can be of mammalian origin, e.g., human.

The ability of the test compound to modulate 52906, 33408, or 12189binding to a compound, e.g., a 52906, 33408, or 12189 substrate, or tobind to 52906, 33408, or 12189 can also be evaluated. This can beaccomplished, for example, by coupling the compound, e.g., thesubstrate, with a radioisotope or enzymatic label such that binding ofthe compound, e.g., the substrate, to 52906, 33408, or 12189 can bedetermined by detecting the labeled compound, e.g., substrate, in acomplex. Alternatively, 52906, 33408, or 12189 could be coupled with aradioisotope or enzymatic label to monitor the ability of a testcompound to modulate 52906, 33408, or 12189 binding to a 52906, 33408,or 12189 substrate in a complex. For example, compounds (e.g., 52906,33408, or 12189 substrates) can be labeled with ¹²⁵I, ³⁵S, ¹⁴C, or ³H,either directly or indirectly, and the radioisotope detected by directcounting of radioemmission or by scintillation counting. Alternatively,compounds can be enzymatically labeled with, for example, horseradishperoxidase, alkaline phosphatase, or luciferase, and the enzymatic labeldetected by determination of conversion of an appropriate substrate toproduct.

The ability of a compound (e.g., a 52906, 33408, or 12189 substrate) tointeract with 52906, 33408, or 12189 with or without the labeling of anyof the interactants can be evaluated. For example, a microphysiometercan be used to detect the interaction of a compound with 52906, 33408,or 12189 without the labeling of either the compound or the 52906,33408, or 12189. McConnell, H. M. et al. (1992) Science 257:1906-1912.As used herein, a “microphysiometer” (e.g., Cytosensor) is ananalytical-instrument that measures the rate at which a cell acidifiesits environment using a light-addressable potentiometric sensor (LAPS).Changes in this acidification rate can be used as an indicator of theinteraction between a compound and 52906, 33408, or 12189.

In yet another embodiment, a cell-free assay is provided in which a52906, 33408, or 12189 protein or biologically active portion thereof iscontacted with a test compound and the ability of the test compound tobind to the 52906, 33408, or 12189 protein or biologically activeportion thereof is evaluated. Preferred biologically active portions ofthe 52906, 33408, or 12189 proteins to be used in assays of the presentinvention include fragments which participate in interactions withnon-52906, 33408, or 12189 molecules, e.g., fragments with high surfaceprobability scores.

Soluble and/or membrane-bound forms of isolated proteins (e.g., 52906,33408, or 12189 proteins or biologically active portions thereof) can beused in the cell-free assays of the invention. When membrane-bound formsof the protein are used, it may be desirable to utilize a solubilizingagent. Examples of such solubilizing agents include non-ionic detergentssuch as n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside,octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton® X-100,Triton® X-114, Thesit®, Isotridecypoly(ethylene glycol ether)_(n),3-[(3-cholamidopropyl)dimethylamminio]-1-propane sulfonate (CHAPS),3-[(3-cholamidopropyl)dimethylamminio]-2-hydroxy-1-propane sulfonate(CHAPSO), or N-dodecyl=N,N-dimethyl-3-ammonio-1-propane sulfonate.

Cell-free assays involve preparing a reaction mixture of the target geneprotein and the test compound under conditions and for a time sufficientto allow the two components to interact and bind, thus forming a complexthat can be removed and/or detected.

The interaction between two molecules can also be detected, e.g., usingfluorescence energy transfer (FET) (see, for example, Lakowicz et al.,U.S. Pat. No. 5,631,169; Stavrianopoulos, et al., U.S. Pat. No.4,868,103). A fluorophore label on the first, ‘donor’ molecule isselected such that its emitted fluorescent energy will be absorbed by afluorescent label on a second, ‘acceptor’ molecule, which in turn isable to fluoresce due to the absorbed energy. Alternately, the ‘donor’protein molecule may simply utilize the natural fluorescent energy oftryptophan residues. Labels are chosen that emit different wavelengthsof light, such that the ‘acceptor’ molecule label may be differentiatedfrom that of the ‘donor’. Since the efficiency of energy transferbetween the labels is related to the distance separating the molecules,the spatial relationship between the molecules can be assessed. In asituation in which binding occurs between the molecules, the fluorescentemission of the ‘acceptor’ molecule label in the assay should bemaximal. An FET binding event can be conveniently measured throughstandard fluorometric detection means well known in the art (e.g., usinga fluorimeter).

In another embodiment, determining the ability of the 52906, 33408, or12189 protein to bind to a target molecule can be accomplished usingreal-time Biomolecular Interaction Analysis (BIA) (see, e.g., Sjolander,S. and Urbaniczky, C. (1991) Anal. Chem. 63:2338-2345 and Szabo et al.(1995) Curr. Opin. Struct. Biol. 5:699-705). “Surface plasmon resonance”or “BIA” detects biospecific interactions in real time, without labelingany of the interactants (e.g., BIAcore). Changes in the mass at thebinding surface (indicative of a binding event) result in alterations ofthe refractive index of light near the surface (the optical phenomenonof surface plasmon resonance (SPR)), resulting in a detectable signalwhich can be used as an indication of real-time reactions betweenbiological molecules.

In one embodiment, the target gene product or the test substance isanchored onto a solid phase. The target gene product/test compoundcomplexes anchored on the solid phase can be detected at the end of thereaction. Preferably, the target gene product can be anchored onto asolid surface, and the test compound, (which is not anchored), can belabeled, either directly or indirectly, with detectable labels discussedherein.

It may be desirable to immobilize either 52906, 33408, or 12189, ananti-52906, 33408, or 12189 antibody or its target molecule tofacilitate separation of complexed from uncomplexed forms of one or bothof the proteins, as well as to accommodate automation of the assay.Binding of a test compound to a 52906, 33408, or 12189 protein, orinteraction of a 52906, 33408, or 12189 protein with a target moleculein the presence and absence of a candidate compound, can be accomplishedin any vessel suitable for containing the reactants. Examples of suchvessels include microtiter plates, test tubes, and micro-centrifugetubes. In one embodiment, a fusion protein can be provided which adds adomain that allows one or both of the proteins to be bound to a matrix.For example, glutathione-S-transferase/52906, 33408, or 12189 fusionproteins or glutathione-S-transferase/target fusion proteins can beadsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis,Mo.) or glutathione derivatized microtiter plates, which are thencombined with the test compound or the test compound and either thenon-adsorbed target protein or 52906, 33408, or 12189 protein, and themixture incubated under conditions conducive to complex formation (e.g.,at physiological conditions for salt and pH). Following incubation, thebeads or microtiter plate wells are washed to remove any unboundcomponents, the matrix immobilized in the case of beads, complexdetermined either directly or indirectly, for example, as describedabove. Alternatively, the complexes can be dissociated from the matrix,and the level of 52906, 33408, or 12189 binding or activity determinedusing standard techniques.

Other techniques for immobilizing either a 52906, 33408, or 12189protein or a target molecule on matrices include using conjugation ofbiotin and streptavidin. Biotinylated 52906, 33408, or 12189 protein ortarget molecules can be prepared from biotin-NHS (N-hydroxy-succinimide)using techniques known in the art (e.g., biotinylation kit, PierceChemicals, Rockford, Ill.), and immobilized in the wells ofstreptavidin-coated 96 well plates (Pierce Chemical).

In order to conduct the assay, the non-immobilized component is added tothe coated surface containing the anchored component. After the reactionis complete, unreacted components are removed (e.g., by washing) underconditions such that any complexes formed will remain immobilized on thesolid surface. The detection of complexes anchored on the solid surfacecan be accomplished in a number of ways. Where the previouslynon-immobilized component is pre-labeled, the detection of labelimmobilized on the surface indicates that complexes were formed. Wherethe previously non-immobilized component is not pre-labeled, an indirectlabel can be used to detect complexes anchored on the surface; e.g.,using a labeled antibody specific for the immobilized component (theantibody, in turn, can be directly labeled or indirectly labeled with,e.g., a labeled anti-Ig antibody).

In one embodiment, this assay is performed utilizing antibodies reactivewith 52906, 33408, or 12189 protein or target molecules but which do notinterfere with binding of the 52906, 33408, or 12189 protein to itstarget molecule. Such antibodies can be derivatized to the wells of theplate, and unbound target or 52906, 33408, or 12189 protein trapped inthe wells by antibody conjugation. Methods for detecting such complexes,in addition to those described above for the GST-immobilized complexes,include immunodetection of complexes using antibodies reactive with the52906, 33408, or 12189 protein or target molecule, as well asenzyme-linked assays which rely on detecting an enzymatic activityassociated with the 52906, 33408, or 12189 protein or target molecule.

Alternatively, cell free assays can be conducted in a liquid phase. Insuch an assay, the reaction products are separated from unreactedcomponents, by any of a number of standard techniques, including but notlimited to: differential centrifugation (see, for example, Rivas, G.,and Minton, A. P., (1993) Trends Biochem Sci 18:284-7); chromatography(gel filtration chromatography, ion-exchange chromatography);electrophoresis (see, e.g., Ausubel, F. et al., eds. Current Protocolsin Molecular Biology 1999, J. Wiley: New York.); and immunoprecipitation(see, for example, Ausubel, F. et al., eds. (1999) Current Protocols inMolecular Biology, J. Wiley: New York). Such resins and chromatographictechniques are known to one skilled in the art (see, e.g., Heegaard, N.H., (1998) J Mol Recognit 11:141-8; Hage, D. S., and Tweed, S. A. (1997)J Chromatogr B Biomed Sci Appl. 699:499-525). Further, fluorescenceenergy transfer may also be conveniently utilized, as described herein,to detect binding without further purification of the complex fromsolution.

In a preferred embodiment, the assay includes contacting the 52906,33408, or 12189 protein or biologically active portion thereof with aknown compound which binds 52906, 33408, or 12189 to form an assaymixture, contacting the assay mixture with a test compound, anddetermining the ability of the test compound to interact with a 52906,33408, or 12189 protein, wherein determining the ability of the testcompound to interact with a 52906, 33408, or 12189 protein includesdetermining the ability of the test compound to preferentially bind to52906, 33408, or 12189 or biologically active portion thereof, or tomodulate the activity of a target molecule, as compared to the knowncompound.

The target gene products of the invention can, in vivo, interact withone or more cellular or extracellular macromolecules, such as proteins.For the purposes of this discussion, such cellular and extracellularmacromolecules are referred to herein as “binding partners.” Compoundsthat disrupt such interactions can be useful in regulating the activityof the target gene product. Such compounds can include, but are notlimited to molecules such as antibodies, peptides, and small molecules.The preferred target genes/products for use in this embodiment are the52906, 33408, or 12189 genes herein identified. In an alternativeembodiment, the invention provides methods for determining the abilityof the test compound to modulate the activity of a 52906, 33408, or12189 protein through modulation of the activity of a downstreameffector of a 52906, 33408, or 12189 target molecule. For example, theactivity of the effector molecule on an appropriate target can bedetermined, or the binding of the effector to an appropriate target canbe determined, as previously described.

To identify compounds that interfere with the interaction between thetarget gene product and its cellular or extracellular bindingpartner(s), a reaction mixture containing the target gene product andthe binding partner is prepared, under conditions and for a timesufficient, to allow the two products to form complex. In order to testan inhibitory agent, the reaction mixture is provided in the presenceand absence of the test compound. The test compound can be initiallyincluded in the reaction mixture, or can be added at a time subsequentto the addition of the target gene and its cellular or extracellularbinding partner. Control reaction mixtures are incubated without thetest compound or with a placebo. The formation of any complexes betweenthe target gene product and the cellular or extracellular bindingpartner is then detected. The formation of a complex in the controlreaction, but not in the reaction mixture containing the test compound,indicates that the compound interferes with the interaction of thetarget gene product and the interactive binding partner. Additionally,complex formation within reaction mixtures containing the test compoundand normal target gene product can also be compared to complex formationwithin reaction mixtures containing the test compound and mutant targetgene product. This comparison can be important in those cases wherein itis desirable to identify compounds that disrupt interactions of mutantbut not normal target gene products.

These assays can be conducted in a heterogeneous or homogeneous format.Heterogeneous assays involve anchoring either the target gene product orthe binding partner onto a solid phase, and detecting complexes anchoredon the solid phase at the end of the reaction. In homogeneous assays,the entire reaction is carried out in a liquid phase. In eitherapproach, the order of addition of reactants can be varied to obtaindifferent information about the compounds being tested. For example,test compounds that interfere with the interaction between the targetgene products and the binding partners, e.g., by competition, can beidentified by conducting the reaction in the presence of the testsubstance. Alternatively, test compounds that disrupt preformedcomplexes, e.g., compounds with higher binding constants that displaceone of the components from the complex, can be tested by adding the testcompound to the reaction mixture after complexes have been formed. Thevarious formats are briefly described below.

In a heterogeneous assay system, either the target gene product or theinteractive cellular or extracellular binding partner, is anchored ontoa solid surface (e.g., a microtiter plate), while the non-anchoredspecies is labeled, either directly or indirectly. The anchored speciescan be immobilized by non-covalent or covalent attachments.Alternatively, an immobilized antibody specific for the species to beanchored can be used to anchor the species to the solid surface.

In order to conduct the assay, the partner of the immobilized species isexposed to the coated surface with or without the test compound. Afterthe reaction is complete, unreacted components are removed (e.g., bywashing) and any complexes formed will remain immobilized on the solidsurface. Where the non-immobilized species is pre-labeled, the detectionof label immobilized on the surface indicates that complexes wereformed. Where the non-immobilized species is not pre-labeled, anindirect label can be used to detect complexes anchored on the surface;e.g., using a labeled antibody specific for the initiallynon-immobilized species (the antibody, in turn, can be directly labeledor indirectly labeled with, e.g., a labeled anti-Ig antibody). Dependingupon the order of addition of reaction components, test compounds thatinhibit complex formation or that disrupt preformed complexes can bedetected.

Alternatively, the reaction can be conducted in a liquid phase in thepresence or absence of the test compound, the reaction productsseparated from unreacted components, and complexes detected; e.g., usingan immobilized antibody specific for one of the binding components toanchor any complexes formed in solution, and a labeled antibody specificfor the other partner to detect anchored complexes. Again, dependingupon the order of addition of reactants to the liquid phase, testcompounds that inhibit complex or that disrupt preformed complexes canbe identified.

In an alternate embodiment of the invention, a homogeneous assay can beused. For example, a preformed complex of the target gene product andthe interactive cellular or extracellular binding partner product isprepared in that either the target gene products or their bindingpartners are labeled, but the signal generated by the label is quencheddue to complex formation (see, e.g., U.S. Pat. No. 4,109,496 thatutilizes this approach for immunoassays). The addition of a testsubstance that competes with and displaces one of the species from thepreformed complex will result in the generation of a signal abovebackground. In this way, test substances that disrupt target geneproduct-binding partner interaction can be identified.

In yet another aspect, the 52906, 33408, or 12189 proteins can be usedas “bait proteins” in a two-hybrid assay or three-hybrid assay (see,e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232;Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al.(1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene8:1693-1696; and Brent WO94/10300), to identify other proteins, whichbind to or interact with 52906, 33408, or 12189 (“52906, 33408, or12189-binding proteins” or “52906, 33408, or 12189-bp”) and are involvedin 52906, 33408, or 12189 activity. Such 52906, 33408, or 12189-bps canbe activators or inhibitors of signals by the 52906, 33408, or 12189proteins or 52906, 33408, or 12189 targets as, for example, downstreamelements of a 52906, 33408, or 12189-mediated signaling pathway.

The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for a 52906, 33408, or12189 protein is fused to a gene encoding the DNA binding domain of aknown transcription factor (e.g., GAL-4). In the other construct, a DNAsequence, from a library of DNA sequences, that encodes an unidentifiedprotein (“prey” or “sample”) is fused to a gene that codes for theactivation domain of the known transcription factor. (Alternatively the:52906, 33408, or 12189 protein can be the fused to the activatordomain.) If the “bait” and the “prey” proteins are able to interact, invivo, forming a 52906, 33408, or 12189-dependent complex, theDNA-binding and activation domains of the transcription factor arebrought into close proximity. This proximity allows transcription of areporter gene (e.g., lacZ) which is operably linked to a transcriptionalregulatory site responsive to the transcription factor. Expression ofthe reporter gene can be detected and cell colonies containing thefunctional transcription factor can be isolated and used to obtain thecloned gene which encodes the protein which interacts with the 52906,33408, or 12189 protein.

In another embodiment, modulators of 52906, 33408, or 12189 expressionare identified. For example, a cell or cell free mixture is contactedwith a candidate compound and the expression of 52906, 33408, or 12189mRNA or protein evaluated relative to the level of expression of 52906,33408, or 12189 mRNA or protein in the absence of the candidatecompound. When expression of 52906, 33408, or 12189 mRNA or protein isgreater in the presence of the candidate compound than in its absence,the candidate compound is identified as a stimulator of 52906, 33408, or12189 mRNA or protein expression. Alternatively, when expression of52906, 33408, or 12189 mRNA or protein is less (statisticallysignificantly less) in the presence of the candidate compound than inits absence, the candidate compound is identified as an inhibitor of52906, 33408, or 12189 mRNA or protein expression. The level of 52906,33408, or 12189 mRNA or protein expression can be determined by methodsdescribed herein for detecting 52906, 33408, or 12189 mRNA or protein.

In another aspect, the invention pertains to a combination of two ormore of the assays described herein. For example, a modulating agent canbe identified using a cell-based or a cell free assay, and the abilityof the agent to modulate the activity of a 52906, 33408, or 12189protein can be confirmed in vivo, e.g., in an animal such as an animalmodel for a disorder characterized by abnormal ion flux such as aneurological disorder or a cardiac disorder.

This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein(e.g., a 52906, 33408, or 12189 modulating agent, an antisense 52906,33408, or 12189 nucleic acid molecule, a 52906, 33408, or 12189-specificantibody, or a 52906, 33408, or 12189-binding partner) in an appropriateanimal model to determine the efficacy, toxicity, side effects, ormechanism of action, of treatment with such an agent. Furthermore, novelagents identified by the above-described screening assays can be usedfor treatments as described herein.

Detection Assays

Portions or fragments of the nucleic acid sequences identified hereincan be used as polynucleotide reagents. For example, these sequences canbe used to: (i) map their respective genes on a chromosome e.g., tolocate gene regions associated with genetic disease or to associate52906, 33408, or 12189 with a disease; (ii) identify an individual froma minute biological sample (tissue typing); and (iii) aid in forensicidentification of a biological sample. These applications are describedin the subsections below.

Chromosome Mapping

The 52906, 33408, or 12189 nucleotide sequences or portions thereof canbe used to map the location of the 52906, 33408, or 12189 genes on achromosome. This process is called chromosome mapping. Chromosomemapping is useful in correlating the 52906, 33408, or 12189 sequenceswith genes associated with disease.

Briefly, 52906, 33408, or 12189 genes can be mapped to chromosomes bypreparing PCR primers (preferably 15-25 bp in length) from the 52906,33408, or 12189 nucleotide sequences. These primers can then be used forPCR screening of somatic cell hybrids containing individual humanchromosomes. Only those hybrids containing the human gene correspondingto the 52906, 33408, or 12189 sequences will yield an amplifiedfragment.

A panel of somatic cell hybrids in which each cell line contains eithera single human chromosome or a small number of human chromosomes, and afull set of mouse chromosomes, can allow easy mapping of individualgenes to specific human chromosomes. (D'Eustachio P. et al. (1983)Science 220:919-924).

Other mapping strategies e.g., in situ hybridization (described in Fan,Y. et al. (1990) Proc. Natl. Acad. Sci. USA, 87:6223-27), pre-screeningwith labeled flow-sorted chromosomes, and pre-selection by hybridizationto chromosome specific cDNA libraries can be used to map 52906, 33408,or 12189 to a chromosomal location.

Fluorescence in situ hybridization (FISH) of a DNA sequence to ametaphase chromosomal spread can further be used to provide a precisechromosomal location in one step. The FISH technique can be used with aDNA sequence as short as 500 or 600 bases. However, clones larger than1,000 bases have a higher likelihood of binding to a unique chromosomallocation with sufficient signal intensity for simple detection.Preferably 1,000 bases, and more preferably 2,000 bases will suffice toget good results at a reasonable amount of time. For a review of thistechnique, see Verma et al., Human Chromosomes: A Manual of BasicTechniques ((1988) Pergamon Press, New York).

Reagents for chromosome mapping can be used individually to mark asingle chromosome or a single site on that chromosome, or panels ofreagents can be used for marking multiple sites and/or multiplechromosomes. Reagents corresponding to noncoding regions of the genesactually are preferred for mapping purposes. Coding sequences are morelikely to be conserved within gene families, thus increasing the chanceof cross hybridizations during chromosomal mapping.

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. (Such data are found, for example, in V.McKusick, Mendelian Inheritance in Man, available on-line through JohnsHopkins University Welch Medical Library). The relationship between agene and a disease, mapped to the same chromosomal region, can then beidentified through linkage analysis (co-inheritance of physicallyadjacent genes), described in, for example, Egeland, J. et al. (1987)Nature, 325:783-787.

Moreover, differences in the DNA sequences between individuals affectedand unaffected with a disease associated with the 52906, 33408, or 12189gene, can be determined. If a mutation is observed in some or all of theaffected individuals but not in any unaffected individuals, then themutation is likely to be the causative agent of the particular disease.Comparison of affected and unaffected individuals generally involvesfirst looking for structural alterations in the chromosomes, such asdeletions or translocations that are visible from chromosome spreads ordetectable using PCR based on that DNA sequence. Ultimately, completesequencing of genes from several individuals can be performed to confirmthe presence of a mutation and to distinguish mutations frompolymorphisms.

Tissue Typing

52906, 33408, or 12189 sequences can be used to identify individualsfrom biological samples using, e.g., restriction fragment lengthpolymorphism (RFLP). In this technique, an individual's genomic DNA isdigested with one or more restriction enzymes, the fragments separated,e.g., in a Southern blot, and probed to yield bands for identification.The sequences of the present invention are useful as additional DNAmarkers for RFLP (described in U.S. Pat. No. 5,272,057).

Furthermore, the sequences of the present invention can also be used todetermine the actual base-by-base DNA sequence of selected portions ofan individual's genome. Thus, the 52906, 33408, or 12189 nucleotidesequences described herein can be used to prepare two PCR primers fromthe 5′ and 3′ ends of the sequences. These primers can then be used toamplify an individual's DNA and subsequently sequence it. Panels ofcorresponding DNA sequences from individuals, prepared in this manner,can provide unique individual identifications, as each individual willhave a unique set of such DNA sequences due to allelic differences.

Allelic variation occurs to some degree in the coding regions of thesesequences, and to a greater degree in the noncoding regions. Each of thesequences described herein can, to some degree, be used as a standardagainst which DNA from an individual can be compared for identificationpurposes. Because greater numbers of polymorphisms occur in thenoncoding regions, fewer sequences are necessary to differentiateindividuals. The noncoding sequences of SEQ ID NO:1, SEQ ID NO:4, or SEQID NO:7 can provide positive individual identification with a panel ofperhaps 10 to 1,000 primers which each. yield a noncoding amplifiedsequence of 100 bases. If predicted coding sequences, such as those inSEQ ID NO:3, SEQ ID NO:6, or SEQ ID NO:7 are used, a more appropriatenumber of primers for positive individual identification would be500-2,000.

If a panel of reagents from 52906, 33408, or 12189 nucleotide sequencesdescribed herein is used to generate a unique identification databasefor an individual, those same reagents can later be used to identifytissue from that individual. Using the unique identification database,positive identification of the individual, living or dead, can be madefrom extremely small tissue samples.

Use of Partial 52906, 33408 or 12189 Sequences in Forensic Biology

DNA-based identification techniques can also be used in forensicbiology. To make such an identification, PCR technology can be used toamplify DNA sequences taken from very small biological samples such astissues, e.g., hair or skin, or body fluids, e.g., blood, saliva, orsemen found at a crime scene. The amplified sequence can then becompared to a standard, thereby allowing identification of the origin ofthe biological sample.

The sequences of the present invention can be used to providepolynucleotide reagents, e.g., PCR primers, targeted to specific loci inthe human genome, which can enhance the reliability of DNA-basedforensic identifications by, for example, providing another“identification marker” (i.e. another DNA sequence that is unique to aparticular individual). As mentioned above, actual base sequenceinformation can be used for identification as an accurate alternative topatterns formed by restriction enzyme generated fragments. Sequencestargeted to noncoding regions of SEQ ID NO:1, SEQ ID NO:4, or SEQ IDNO:7 (e.g., fragments derived from the noncoding regions of SEQ ID NO:1,SEQ ID NO:4, or SEQ ID NO:7 having a length of at least 20 bases,preferably at least 30 bases) are particularly appropriate for this use.

The 52906, 33408, or 12189 nucteotide sequences described herein canfurther be used to provide polynucleotide reagents, e.g., labeled orlabelable probes which can be used in, for example, an in situhybridization technique, to identify a specific tissue. This can be veryuseful in cases where a forensic pathologist is presented with a tissueof unknown origin. Panels of such 52906, 33408, or 12189 probes can beused to identify tissue by species and/or by organ type.

In a similar fashion, these reagents, e.g., 52906, 33408, or 12189primers or probes can be used to screen tissue culture for contamination(i.e. screen for the presence of a mixture of different types of cellsin a culture).

Predictive Medicine

The present invention also pertains to the field of predictive medicinein which diagnostic assays, prognostic assays, and monitoring clinicaltrials are used for prognostic (predictive) purposes to thereby treat anindividual.

Generally, the invention provides, a method of determining if a subjectis at risk for a disorder related to a lesion in or the misexpression ofa gene which encodes 52906, 33408, or 12189.

Such disorders include, e.g., a disorder associated with themisexpression of 52906, 33408, or 12189 gene, or a disordercharacterized by abnormal ion flux such as a neurological disorder or acardiac disorder.

The method includes one or more of the following:

detecting, in a tissue of the subject, the presence or absence of amutation which affects the expression of the 52906, 33408, or 12189gene, or detecting the presence or absence of a mutation in a regionwhich controls the expression of the gene, e.g., a mutation in the 5′control region;

detecting, in a tissue of the subject, the presence or absence of amutation which alters the structure of the 52906, 33408, or 12189 gene;

detecting, in a tissue of the subject, the misexpression of the 52906,33408, or 12189 gene, at the mRNA level, e.g., detecting a non-wild typelevel of a mRNA;

detecting, in a tissue of the subject, the misexpression of the gene, atthe protein level, e.g., detecting a non-wild type level of a 52906,33408, or 12189 polypeptide.

In preferred embodiments the method includes: ascertaining the existenceof at least one of: a deletion of one or more nucleotides from the52906, 33408, or 12189 gene; an insertion of one or more nucleotidesinto the gene, a point mutation, e.g., a substitution of one or morenucleotides of the gene, a gross chromosomal rearrangement of the gene,e.g., a translocation, inversion, or deletion.

For example, detecting the genetic lesion can include: (i) providing aprobe/primer including an oligonucleotide containing a region ofnucleotide sequence which hybridizes to a sense or antisense sequencefrom SEQ ID NO:1, SEQ ID NO:4, or SEQ ID NO:7, or naturally occurringmutants thereof or 5′ or 3′ flanking sequences naturally associated withthe 52906, 33408, or 12189 gene; (ii) exposing the probe/primer tonucleic acid of the tissue; and detecting, by hybridization, e.g., insitu hybridization, of the probe/primer to the nucleic acid, thepresence or absence of the genetic lesion.

In preferred embodiments detecting the misexpression includesascertaining the existence of at least one of: an alteration in thelevel of a messenger RNA transcript of the 52906, 33408, or 12189 gene;the presence of a non-wild type splicing pattern of a messenger RNAtranscript of the gene; or a non-wild type level of 52906, 33408, or12189.

Methods of the invention can be used prenatally or to determine if asubject's offspring will be at risk for a disorder.

In preferred embodiments the method includes determining the structureof a 52906, 33408, or 12189 gene, an abnormal structure being indicativeof risk for the disorder.

In preferred embodiments the method includes contacting a sample fromthe subject with an antibody to the 52906, 33408, or 12189 protein or anucleic acid, which hybridizes specifically with the gene. These andother embodiments are discussed below.

Diagnostic and Prognostic Assays

Diagnostic and prognostic assays of the invention include methods forassessing the expression level of 52906, 33408, or 12189 molecules andfor identifying variations and mutations in the sequence of 52906,33408, or 12189 molecules.

Expression Monitoring and Profiling

The presence, level, or absence of 52906, 33408, or 12189 protein ornucleic acid in a biological sample can be evaluated by obtaining abiological sample from a test subject and contacting the biologicalsample with a compound or an agent capable of detecting 52906, 33408, or12189 protein or nucleic acid (e.g., mRNA, genomic DNA) that encodes52906, 33408, or 12189 protein such that the presence of 52906, 33408,or 12189 protein or nucleic acid is detected in the biological sample.The term “biological sample” includes tissues, cells and biologicalfluids isolated from a subject, as well as tissues, cells and fluidspresent within a subject. A preferred biological sample is serum. Thelevel of expression of the 52906, 33408, or 12189 gene can be measuredin a number of ways, including, but not limited to: measuring the mRNAencoded by the 52906, 33408, or 12189 genes; measuring the amount ofprotein encoded by the 52906, 33408, or 12189 genes; or measuring theactivity of the protein encoded by the 52906, 33408, or 12189 genes.

The level of mRNA corresponding to the 52906, 33408, or 12189 gene in acell can be determined both by in situ and by in vitro formats.

The isolated mRNA can be used in hybridization or amplification assaysthat include, but are not limited to, Southern or Northern analyses,polymerase chain reaction analyses and probe arrays. One preferreddiagnostic method for the detection of mRNA levels involves contactingthe isolated mRNA with a nucleic acid molecule (probe) that canhybridize to the mRNA encoded by the gene being detected. The nucleicacid probe can be, for example, a full-length 52906, 33408, or 12189nucleic acid, such as the nucleic acid of SEQ ID NO:1, SEQ ID NO:4, orSEQ ID NO:7, or a portion thereof, such as an oligonucleotide of atleast 7, 15, 30, 50, 100, 250 or 500 nucleotides in length andsufficient to specifically hybridize under stringent conditions to52906, 33408, or 12189 mRNA or genomic DNA. The probe can be disposed onan address of an array, e.g., an array described below. Other suitableprobes for use in the diagnostic assays are described herein.

In one format, mRNA (or cDNA) is immobilized on a surface and contactedwith the probes, for example by running the isolated mRNA on an agarosegel and transferring the mRNA from the gel to a membrane, such asnitrocellulose. In an alternative format, the probes are immobilized ona surface and the mRNA (or cDNA) is contacted with the probes, forexample, in a two-dimensional gene chip array described below. A skilledartisan can adapt known mRNA detection methods for use in detecting thelevel of mRNA encoded by the 52906, 33408, or 12189 genes.

The level of mRNA in a sample that is encoded by one of 52906, 33408, or12189 can be evaluated with nucleic acid amplification, e.g., by rtPCR(Mullis (1987) U.S. Pat. No. 4,683,202), ligase chain reaction (Barany(1991) Proc. Natl. Acad. Sci. USA 88:189-193), self sustained sequencereplication (Guatelli et al., (1990) Proc. Natl. Acad. Sci. USA87:1874-1878), transcriptional amplification system (Kwoh et al.,(1989), Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase(Lizardi et al., (1988) Bio/Technology 6:1197), rolling circlereplication (Lizardi et al., U.S. Pat. No. 5,854,033) or any othernucleic acid amplification method, followed by the detection of theamplified molecules using techniques known in the art. As used herein,amplification primers are defined as being a pair of nucleic acidmolecules that can anneal to 5′ or 3′ regions of a gene (plus and minusstrands, respectively, or vice-versa) and contain a short region inbetween. In general, amplification primers are from about 10 to 30nucleotides in length and flank a region from about 50 to 200nucleotides in length. Under appropriate conditions and with appropriatereagents, such primers permit the amplification of a nucleic acidmolecule comprising the nucleotide sequence flanked by the primers.

For in situ methods, a cell or tissue sample can be prepared/processedand immobilized on a support, typically a glass slide, and thencontacted with a probe that can hybridize to mRNA that encodes the52906, 33408, or 12189 gene being analyzed.

In another embodiment, the methods further contacting a control samplewith a compound or agent capable of detecting 52906, 33408, or 12189mRNA, or genomic DNA, and comparing the presence of 52906, 33408, or12189 mRNA or genomic DNA in the control sample with the presence of52906, 33408, or 12189 mRNA or genomic DNA in the test sample. In stillanother embodiment, serial analysis of gene expression, as described inU.S. Pat. No. 5,695,937, is used to detect 52906, 33408, or 12189transcript levels.

A variety of methods can be used to determine the level of proteinencoded by 52906, 33408, or 12189. In general, these methods includecontacting an agent that selectively binds to the protein, such as anantibody with a sample, to evaluate the level of protein in the sample.In a preferred embodiment, the antibody bears a detectable label.Antibodies can be polyclonal, or more preferably, monoclonal. An intactantibody, or a fragment thereof (e.g., Fab or F(ab′)₂) can be used. Theterm “labeled”, with regard to the probe or antibody, is intended toencompass direct labeling of the probe or antibody by coupling (i.e.,physically linking) a detectable substance to the probe or antibody, aswell as indirect labeling of the probe or antibody by reactivity with adetectable substance. Examples of detectable substances are providedherein.

The detection methods can be used to detect 52906, 33408, or 12189protein in a biological sample in vitro as well as in vivo. In vitrotechniques for detection of 52906, 33408, or 12189 protein includeenzyme linked immunosorbent assays (ELISAs), immunoprecipitations,immunofluorescence, enzyme immunoassay (EIA), radioimmunoassay (RIA),and Western blot analysis. In vivo techniques for detection of 52906,33408, or 12189 protein include introducing into a subject a labeledanti-52906, 33408, or 12189 antibody. For example, the antibody can belabeled with a radioactive marker whose presence and location in asubject can be detected by standard imaging techniques. In anotherembodiment, the sample is labeled, e.g., biotinylated and then contactedto the antibody, e.g., an anti-52906, 33408, or 12189 antibodypositioned on an antibody array (as described below). The sample can bedetected, e.g., with avidin coupled to a fluorescent label.

In another embodiment, the methods further include contacting thecontrol sample with a compound or agent capable of detecting 52906,33408, or 12189 protein, and comparing the presence of 52906, 33408, or12189 protein in the control sample with the presence of 52906, 33408,or 12189 protein in the test sample.

The invention also includes kits for detecting the presence of 52906,33408, or 12189 in a biological sample. For example, the kit can includea compound or agent capable of detecting 52906, 33408, or 12189 proteinor mRNA in a biological sample; and a standard. The compound or agentcan be packaged in a suitable container. The kit can further compriseinstructions for using the kit to detect 52906, 33408, or 12189 proteinor nucleic acid.

For antibody-based kits, the kit can include: (1) a first antibody(e.g., attached to a solid support) which binds to a polypeptidecorresponding to a marker of the invention; and, optionally, (2) asecond, different antibody which binds to either the polypeptide or thefirst antibody and is conjugated to a detectable agent.

For oligonucleotide-based kits, the kit can include: (1) anoligonucleotide, e.g., a detectably labeled oligonucleotide, whichhybridizes to a nucleic acid sequence encoding a polypeptidecorresponding to a marker of the invention or (2) a pair of primersuseful for amplifying a nucleic acid molecule corresponding to a markerof the invention. The kit can also includes a buffering agent, apreservative, or a protein stabilizing agent. The kit can also includescomponents necessary for detecting the detectable agent (e.g., an enzymeor a substrate). The kit can also contain a control sample or a seriesof control samples which can be assayed and compared to the test samplecontained. Each component of the kit can be enclosed within anindividual container and all of the various containers can be within asingle package, along with instructions for interpreting the results ofthe assays performed using the kit.

The diagnostic methods described herein can identify.subjects having, orat risk of developing, a disease or disorder associated withmisexpressed or aberrant or unwanted 52906, 33408, or 12189 expressionor activity. As used herein, the term “unwanted” includes an unwantedphenomenon involved in a biological response such disorderscharacterized by abnormal ion flux such as neurological disorders orcardiac disorders.

In one embodiment, a disease or disorder associated with aberrant orunwanted 52906, 33408, or 12189 expression or activity is identified. Atest sample is obtained from a subject and 52906, 33408, or 12189protein or nucleic acid (e.g., mRNA or genomic DNA) is evaluated,wherein the level, e.g., the presence or absence, of 52906, 33408, or12189 protein or nucleic acid is diagnostic for a subject having or atrisk of developing a disease or disorder associated with aberrant orunwanted 52906, 33408, or 12189 expression or activity. As used herein,a “test sample” refers to a biological sample obtained from a subject ofinterest, including a biological fluid (e.g., serum), cell sample, ortissue.

The prognostic assays described herein can be used to determine whethera subject can be administered an agent (e.g., an agonist, antagonist,peptidomimetic, protein, peptide, nucleic acid, small molecule, or otherdrug candidate) to treat a disease or disorder associated with aberrantor unwanted 52906, 33408, or 12189 expression or activity. For example,such methods can be used to determine whether a subject can beeffectively treated with an agent for disorders characterized byabnormal ion flux such as neurological disorders or cardiac disorders.

In another aspect, the invention features a computer medium having aplurality of digitally encoded data records. Each data record includes avalue representing the level of expression of 52906, 33408, or 12189 ina sample, and a descriptor of the sample. The descriptor of the samplecan be an identifier of the sample, a subject from which the sample wasderived (e.g., a patient), a diagnosis, or a treatment (e.g., apreferred treatment). In a preferred embodiment, the data record furtherincludes values representing the level of expression of genes other than52906, 33408, or 12189 (e.g., other genes associated with a 52906,33408, or 12189-disorder, or other genes on an array). The data recordcan be structured as a table, e.g., a table that is part of a databasesuch as a relational database (e.g., a SQL database of the Oracle orSybase database environments).

Also featured is a method of evaluating a sample. The method includesproviding a sample, e.g., from the subject, and determining a geneexpression profile of the sample, wherein the profile includes a valuerepresenting the level of 52906, 33408, or 12189 expression. The methodcan further include comparing the value or the profile (i.e., multiplevalues) to a reference value or reference profile. The gene expressionprofile of the sample can be obtained by any of the methods describedherein (e.g., by providing a nucleic acid from the sample and contactingthe nucleic acid to an array). The method can be used to diagnose an ionflux-related disorder in a subject wherein a modulation (increase ordecrease) in 52906, 33408, or 12189 expression is an indication that thesubject has or is disposed to having a disorder characterized byabnormal ion flux such as a neurological disorder or a cardiac disorder.The method can be used to monitor a treatment for an ion flux-relateddisorder in a subject. For example, the gene expression profile can bedetermined for a sample from a subject undergoing treatment. The profilecan be compared to a reference profile or to a profile obtained from thesubject prior to treatment or prior to onset of the disorder (see, e.g.,Golub et al. (1999) Science 286:531).

In yet another aspect, the invention features a method of evaluating atest compound (see also, “Screening Assays”, above). The method includesproviding a cell and a test compound; contacting the test compound tothe cell; obtaining a subject expression profile for the contacted cell;and comparing the subject expression profile to one or more referenceprofiles. The profiles include a value representing the level of 52906,33408, or 12189 expression. In a preferred embodiment, the subjectexpression profile is compared to a target profile, e.g., a profile fora normal cell or for desired condition of a cell. The test compound isevaluated favorably if the subject expression profile is more similar tothe target profile than an expression profile obtained from anuncontacted cell.

In another aspect, the invention features, a method of evaluating asubject. The method includes: a) obtaining a sample from a subject,e.g., from a caregiver, e.g., a caregiver who obtains the sample fromthe subject; b) determining a subject expression profile for the sample.Optionally, the method further includes either or both of steps: c)comparing the subject expression profile to one or more referenceexpression profiles; and d) selecting the reference profile most similarto the subject reference profile. The subject expression profile and thereference profiles include a value representing the level of 52906,33408, or 12189 expression. A variety of routine statistical measurescan be used to compare two reference profiles. One possible metric isthe length of the distance vector that is the difference between the twoprofiles. Each of the subject and reference profile is represented as amulti-dimensional vector, wherein each dimension is a value in theprofile.

The method can further include transmitting a result to a caregiver. Theresult can be the subject expression profile, a result of a comparisonof the subject expression profile with another profile, a most similarreference profile, or a descriptor of any of the aforementioned. Theresult can be transmitted across a computer network, e.g., the resultcan be in the form of a computer transmission, e.g., a computer datasignal embedded in a carrier wave.

Also featured is a computer medium having executable code for effectingthe following steps: receive a subject expression profile; access adatabase of reference expression profiles; and either i) select amatching reference profile most similar to the subject expressionprofile or ii) determine at least one comparison score for thesimilarity of the subject expression profile to at least one referenceprofile. The subject expression profile, and the reference expressionprofiles each include a value representing the level of 52906, 33408, or12189 expression.

Arrays and Uses Thereof

In another aspect, the invention features an array that includes asubstrate having a plurality of addresses. At least one address of theplurality includes a capture probe that binds specifically to a 52906,33408, or 12189 molecule (e.g., a 52906, 33408, or 12189 nucleic acid ora 52906, 33408, or 12189 polypeptide). The array can have a density ofat least than 10, 50, 100, 200, 500, 1,000, 2,000, or 10,000 or moreaddresses/cm², and ranges between. In a preferred embodiment, theplurality of addresses includes at least 10, 100, 500, 1,000, 5,000,10,000, 50,000 addresses. In a preferred embodiment, the plurality ofaddresses includes equal to or less than 10, 100, 500, 1,000, 5,000,10,000, or 50,000 addresses. The substrate can be a two-dimensionalsubstrate such as a glass slide, a wafer (e.g., silica or plastic), amass spectroscopy plate, or a three-dimensional substrate such as a gelpad. Addresses in addition to address of the plurality can be disposedon the array.

In a preferred embodiment, at least one address of the pluralityincludes a nucleic acid capture probe that hybridizes specifically to a52906, 33408, or 12189 nucleic acid, e.g., the sense or anti-sensestrand. In one preferred embodiment, a subset of addresses of theplurality of addresses has a nucleic acid capture probe for 52906,33408, or 12189. Each address of the subset can include a capture probethat hybridizes to a different region of a 52906, 33408, or 12189nucleic acid. In another preferred embodiment, addresses of the subsetinclude a capture probe for a 52906, 33408, or 12189 nucleic acid. Eachaddress of the subset is unique, overlapping, and complementary to adifferent variant of 52906, 33408, or 12189 (e.g., an allelic variant,or all possible hypothetical variants). The array can be used tosequence 52906, 33408, or 12189 by hybridization (see, e.g., U.S. Pat.No. 5,695,940).

An array can be generated by various methods, e.g., by photolithographicmethods (see, e.g., U.S. Pat. Nos. 5,143,854; 5,510,270; and 5,527,681),mechanical methods (e.g., directed-flow methods as described in U.S.Pat. No. 5,384,261), pin-based methods (e.g., as described in U.S. Pat.No. 5,288,514), and bead-based techniques (e.g., as described in PCTUS/93/04145).

In another preferred embodiment, at least one address of the pluralityincludes a polypeptide capture probe that binds specifically to a 52906,33408, or 12189 polypeptide or fragment thereof. The polypeptide can bea naturally-occurring interaction partner of 52906, 33408, or 12189polypeptide. Preferably, the polypeptide is an antibody, e.g., anantibody described herein (see “Anti-52906, 33408, or 12189 Antibodies,”above), such as a monoclonal antibody or a single-chain antibody.

In another aspect, the invention features a method of analyzing theexpression of 52906, 33408, or 12189. The method includes providing anarray as described above; contacting the array with a sample anddetecting binding of a 52906, 33408, or 12189-molecule (e.g., nucleicacid or polypeptide) to the array. In a preferred embodiment, the arrayis a nucleic acid array. Optionally the method further includesamplifying nucleic acid from the sample prior or during contact with thearray.

In another embodiment, the array can be used to assay gene expression ina tissue to ascertain tissue specificity of genes in the array,particularly the expression of 52906, 33408, or 12189. If a sufficientnumber of diverse samples is analyzed, clustering (e.g., hierarchicalclustering, k-means clustering, Bayesian clustering and the like) can beused to identify other genes which are co-regulated with 52906, 33408,or 12189. For example, the array can be used for the quantitation of theexpression of multiple genes. Thus, not only tissue specificity, butalso the level of expression of a battery of genes in the tissue isascertained. Quantitative data can be used to group (e.g., cluster)genes on the basis of their tissue expression per se and level ofexpression in that tissue.

For example, array analysis of gene expression can be used to assess theeffect of cell-cell interactions on 52906, 33408, or 12189 expression. Afirst tissue can be perturbed and nucleic acid from a second tissue thatinteracts with the first tissue can be analyzed. In this context, theeffect of one cell type on another cell type in response to a biologicalstimulus can be determined, e.g., to monitor the effect of cell-cellinteraction at the level of gene expression.

In another embodiment, cells are contacted with a therapeutic agent. Theexpression profile of the cells is determined using the array, and theexpression profile is compared to the profile of like cells notcontacted with the agent. For example, the assay can be used todetermine or analyze the molecular basis of an undesirable effect of thetherapeutic agent. If an agent is administered therapeutically to treatone cell type but has an undesirable effect on another cell type, theinvention provides an assay to determine the molecular basis of theundesirable effect and thus provides the opportunity to co-administer acounteracting agent or otherwise treat the undesired effect. Similarly,even within a single cell type, undesirable biological effects can bedetermined at the molecular level. Thus, the effects of an agent onexpression of other than the target gene can be ascertained andcounteracted.

In another embodiment, the array can be used to monitor expression ofone or more genes in the array with respect to time. For example,samples obtained from different time points can be probed with thearray. Such analysis can identify and/or characterize the development ofa 52906, 33408, or 12189-associated disease or disorder; and processes,such as a cellular transformation associated with a 52906, 33408, or12189-associated disease or disorder. The method can also evaluate thetreatment and/or progression of a 52906, 33408, or 12189-associateddisease or disorder

The array is also useful for ascertaining differential expressionpatterns of one or more genes in normal and abnormal cells. Thisprovides a battery of genes (e.g., including 52906, 33408, or 12189 )that could serve as a molecular target for diagnosis or therapeuticintervention.

In another aspect, the invention features an array having a plurality ofaddresses. Each address of the plurality includes a unique polypeptide.At least one address of the plurality has disposed thereon a 52906,33408, or 12189 polypeptide or fragment thereof. Methods of producingpolypeptide arrays are described in the art, e.g., in De Wildt et al.(2000). Nature Biotech. 18, 989-994; Lueking et al. (1999). Anal.Biochem. 270, 103-111; Ge, H. (2000). Nucleic Acids Res. 28, e3, I-VII;MacBeath, G., and Schreiber, S. L. (2000). Science 289, 1760-1763; andWO 99/51773A1. In a preferred embodiment, each. addresses of theplurality has disposed thereon a polypeptide at least 60, 70, 80,85, 90,95 or 99% identical to a 52906, 33408, or 12189 polypeptide or fragmentthereof. For example, multiple variants of a 52906, 33408, or 12189polypeptide (e.g., encoded by allelic variants, site-directed mutants,random mutants, or combinatorial mutants) can be disposed at individualaddresses of the plurality. Addresses in addition to the address of theplurality can be disposed on the array.

The polypeptide array can be used to detect a 52906, 33408, or 12189binding compound, e.g., an antibody in a sample from a subject withspecificity for a 52906, 33408, or 12189 polypeptide or the presence ofa 52906, 33408, or 12189-binding protein or ligand.

The array is also useful for ascertaining the effect of the expressionof a gene on the expression of other genes in the same cell or indifferent cells (e.g., ascertaining the effect of 52906, 33408, or 12189expression on the expression of other genes). This provides, forexample, for a selection of alternate molecular targets for therapeuticintervention if the ultimate or downstream target cannot be regulated.

In another aspect, the invention features a method of analyzing aplurality of probes. The method is useful, e.g., for analyzing geneexpression. The method includes: providing a two dimensional arrayhaving a plurality of addresses, each address of the plurality beingpositionally distinguishable from each other address of the pluralityhaving a unique capture probe, e.g., wherein the capture probes are froma cell or subject which express 52906, 33408, or 12189 or from a cell orsubject in which a 52906, 33408, or 12189 mediated response has beenelicited, e.g., by contact of the cell with 52906, 33408, or 12189nucleic acid or protein, or administration to the cell or subject 52906,33408, or 12189 nucleic acid or protein; providing a two dimensionalarray having a plurality of addresses, each address of the pluralitybeing positionally distinguishable from each other address of theplurality, and each address of the plurality having a unique captureprobe, e.g., wherein the capture probes are from a cell or subject whichdoes not express 52906, 33408, or 12189 (or does not express as highlyas in the case of the 52906, 33408, or 12189 positive plurality ofcapture probes) or from a cell or subject which in which a 52906, 33408,or 12189 mediated response has not been elicited (or has been elicitedto a lesser extent than in the first sample); contacting the array withone or more inquiry probes (which is preferably other than a 52906,33408, or 12189 nucleic acid, polypeptide, or antibody), and therebyevaluating the plurality of capture probes. Binding, e.g., in the caseof a nucleic acid, hybridization with a capture probe at an address ofthe plurality, is detected, e.g., by signal generated from a labelattached to the nucleic acid, polypeptide, or antibody.

In another aspect, the invention features a method of analyzing aplurality of probes or a sample. The method is useful, e.g., foranalyzing gene expression. The method includes: providing a twodimensional array having a plurality of addresses, each address of theplurality being positionally distinguishable from each other address ofthe plurality having a unique capture probe, contacting the array with afirst sample from a cell or subject which express or mis-express 52906,33408, or 12189 or from a cell or subject in which a 52906, 33408, or12189-mediated response has been elicited, e.g., by contact of the cellwith 52906, 33408, or 12189 nucleic acid or protein, or administrationto the cell or subject 52906, 33408, or 12189 nucleic acid or protein;providing a two dimensional array having a plurality of addresses, eachaddress of the plurality being positionally distinguishable from eachother address of the plurality, and each address of the plurality havinga unique capture probe, and contacting the array with a second samplefrom a cell or subject which does not express 52906, 33408, or 12189 (ordoes not express as highly as in the case of the 52906, 33408, or 12189positive plurality of capture probes) or from a cell or subject which inwhich a 52906, 33408, or 12189 mediated response has not been elicited(or has been elicited to a lesser extent than in the first sample); andcomparing the binding of the first sample with the binding of the secondsample. Binding, e.g., in the case of a nucleic acid, hybridization witha capture probe at an address of the plurality, is detected, e.g., bysignal generated from a label attached to the nucleic acid, polypeptide,or antibody. The same array can be used for both samples or differentarrays can be used. If different arrays are used the plurality ofaddresses with capture probes should be present on both arrays.

In another aspect, the invention features a method of analyzing 52906,33408, or 12189, e.g., analyzing structure, function, or relatedness toother nucleic acid or amino acid sequences. The method includes:providing a 52906, 33408, or 12189 nucleic acid or amino acid sequence;comparing the 52906, 33408, or 12189 sequence with one or morepreferably a plurality of sequences from a collection of sequences,e.g., a nucleic acid or protein sequence database; to thereby analyze52906, 33408, or 12189.

Detection of Variations or Mutations

The methods of the invention can also be used to detect geneticalterations in a 52906, 33408, or 12189 gene, thereby determining if asubject with the altered gene is at risk for a disorder characterized bymisregulation in 52906, 33408, or 12189 protein activity or nucleic acidexpression, such as a disorder characterized by abnormal ion flux suchas a neurological disorder or a cardiac disorder. In preferredembodiments, the methods include detecting, in a sample from thesubject, the presence or absence of a genetic alteration characterizedby at least one of an alteration affecting the integrity of a geneencoding a 52906, 33408, or 12189-protein, or the mis-expression of the52906, 33408, or 12189 gene. For example, such genetic alterations canbe detected by ascertaining the existence of at least one of 1) adeletion of one or more nucleotides from a 52906, 33408, or 12189 gene;2) an addition of one or more nucleotides to a 52906, 33408, or 12189gene; 3) a substitution of one or more nucleotides of a 52906, 33408, or12189 gene, 4) a chromosomal rearrangement of a 52906, 33408, or 12189gene; 5) an alteration in the level of a messenger RNA transcript of a52906, 33408, or 12189 gene, 6) aberrant modification of a 52906, 33408,or 12189 gene, such as of the methylation pattern of the genomic DNA, 7)the presence of a non-wild type splicing pattern of a messenger RNAtranscript of a 52906, 33408, or 12189 gene, 8) a non-wild type level ofa 52906, 33408, or 12189-protein, 9) allelic loss of a 52906, 33408, or12189 gene, and 10) inappropriate post-translational modification of a52906, 33408, or 12189-protein.

An alteration can be detected without a probe/primer in a polymerasechain reaction, such as anchor PCR or RACE PCR, or, alternatively, in aligation chain reaction (LCR), the latter of which can be particularlyuseful for detecting point mutations in the 52906, 33408, or 12189-gene.This method can include the steps of collecting a sample of cells from asubject, isolating nucleic acid (e.g., genomic, mRNA or both) from thesample, contacting the nucleic acid sample with one or more primerswhich specifically hybridize to a 52906, 33408, or 12189 gene underconditions such that hybridization and amplification of the 52906,33408, or 12189-gene (if present) occurs, and detecting the presence orabsence of an amplification product, or detecting the size of theamplification product and comparing the length to a control sample. Itis anticipated that PCR and/or LCR may be desirable to use as apreliminary amplification step in conjunction with any of the techniquesused for detecting mutations described herein. Alternatively, otheramplification methods described herein or known in the art can be used.

In another embodiment, mutations in a 52906, 33408, or 12189 gene from asample cell can be identified by detecting alterations in restrictionenzyme cleavage patterns. For example, sample and control DNA isisolated, amplified (optionally), digested with one or more restrictionendonucleases, and fragment length sizes are determined, e.g., by gelelectrophoresis and compared. Differences in fragment length sizesbetween sample and control DNA indicates mutations in the sample DNA.Moreover, the use of sequence specific ribozymes (see, for example, U.S.Pat. No. 5,498,531) can be used to score for the presence of specificmutations by development or loss of a ribozyme cleavage site.

In other embodiments, genetic mutations in 52906, 33408, or 12189 can beidentified by hybridizing a sample and control nucleic acids, e.g., DNAor RNA, two-dimensional arrays, e.g., chip based arrays. Such arraysinclude a plurality of addresses, each of which is positionallydistinguishable from the other. A different probe is located at eachaddress of the plurality. A probe can be complementary to a region of a52906, 33408, or 12189 nucleic acid or a putative variant (e.g., allelicvariant) thereof. A probe can have one or more mismatches to a region ofa 52906, 33408, or 12189 nucleic acid (e.g., a destabilizing mismatch).The arrays can have a high density of addresses, e.g., can containhundreds or thousands of oligonucleotides probes (Cronin, M. T. et al.(1996). Human Mutation 7: 244-255; Kozal, M. J. et al. (1996) NatureMedicine 2: 753-759). For example, genetic mutations in 52906, 33408, or12189 can be identified in two-dimensional arrays containinglight-generated DNA probes as described in Cronin, M. T. et al. supra.Briefly, a first hybridization array of probes can be used to scanthrough long stretches of DNA in a sample and control to identify basechanges between the sequences by making linear arrays of sequentialoverlapping probes. This step allows the identification of pointmutations. This step is followed by a second hybridization array thatallows the characterization of specific mutations by using smaller,specialized probe arrays complementary to all variants or mutationsdetected. Each mutation array is composed of parallel probe sets, onecomplementary to the wild-type gene and the other complementary to themutant gene.

In yet another embodiment, any of a variety of sequencing reactionsknown in the art can be used to directly sequence the 52906, 33408, or12189 gene and detect mutations by comparing the sequence of the sample52906, 33408, or 12189 with the corresponding wild-type (control)sequence. Automated sequencing procedures can be utilized whenperforming the diagnostic assays ((1995) Biotechniques 19:448),including sequencing by mass spectrometry.

Other methods for detecting mutations in the 52906, 33408, or 12189 geneinclude methods in which protection from cleavage agents is used todetect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers etal. (1985) Science 230:1242; Cotton et al. (1988) Proc. Natl Acad SciUSA 85:4397; Saleeba et al. (1992) Methods Enzymol. 217:286-295).

In still another embodiment, the mismatch cleavage reaction employs oneor more proteins that recognize mismatched base pairs in double-strandedDNA (so called “DNA mismatch repair” enzymes) in defined systems fordetecting and mapping point mutations in 52906, 33408, or 12189 cDNAsobtained from samples of cells. For example, the mutY enzyme of E. colicleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLacells cleaves T at G/T mismatches (Hsu et al. (1994) Carcinogenesis15:1657-1662; U.S. Pat. No. 5,459,039).

In other embodiments, alterations in electrophoretic mobility will beused to identify mutations in 52906, 33408, or 12189 genes. For example,single strand conformation polymorphism (SSCP) may be used to detectdifferences in electrophoretic mobility between mutant and wild typenucleic acids (Orita et al. (1989) Proc Natl. Acad. Sci USA: 86:2766,see also Cotton (1993) Mutat. Res. 285:125-144; and Hayashi (1992)Genet. Anal. Tech. Appl. 9:73-79). Single-stranded DNA fragments ofsample and control 52906, 33408, or 12189 nucleic acids will bedenatured and allowed to renature. The secondary structure ofsingle-stranded nucleic acids varies according to sequence, theresulting alteration in electrophoretic mobility enables the detectionof even a single base change. The DNA fragments may be labeled ordetected with labeled probes. The sensitivity of the assay may beenhanced by using RNA (rather than DNA), in which the secondarystructure is more sensitive to a change in sequence. In a preferredembodiment, the subject method utilizes heteroduplex analysis toseparate double stranded heteroduplex molecules on the basis of changesin electrophoretic mobility (Keen et al. (1991) Trends Genet 7:5).

In yet another embodiment, the movement of mutant or wild-type fragmentsin polyacrylamide gels containing a gradient of denaturant is assayedusing denaturing gradient gel electrophoresis (DGGE) (Myers et al.(1985) Nature 313:495). When DGGE is used as the method of analysis, DNAwill be modified to insure that it does not completely denature, forexample by adding a GC clamp of approximately 40 bp of high-meltingGC-rich DNA by PCR. In a further embodiment, a temperature gradient isused in place of a denaturing gradient to identify differences in themobility of control and sample DNA (Rosenbaum and Reissner (1987)Biophys Chem 265:12753).

Examples of other techniques for detecting point mutations include, butare not limited to, selective oligonucleotide hybridization, selectiveamplification, or selective primer extension (Saiki et al. (1986) Nature324:163); Saiki et al. (1989) Proc. Natl Acad. Sci USA 86:6230). Afurther method of detecting point mutations is the chemical ligation ofoligonucleotides as described in Xu et al. ((2001) Nature Biotechnol.19:148). Adjacent oligonucleotides, one of which selectively anneals tothe query site, are ligated together if the nucleotide at the query siteof the sample nucleic acid is complementary to the queryoligonucleotide; ligation can be monitored, e.g., by fluorescent dyescoupled to the oligonucleotides.

Alternatively, allele specific amplification technology that depends onselective PCR amplification may be used in conjunction with the instantinvention. Oligonucleotides used as primers for specific amplificationmay carry the mutation of interest in the center of the molecule (sothat amplification depends on differential hybridization) (Gibbs et al.(1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3′ end of oneprimer where, under appropriate conditions, mismatch can prevent, orreduce polymerase extension (Prossner (1993) Tibtech 11:238). Inaddition it may be desirable to introduce a novel restriction site inthe region of the mutation to create cleavage-based detection (Gaspariniet al. (1992) Mol. Cell Probes 6:1). It is anticipated that in certainembodiments amplification may also be performed using Taq ligase foramplification (Barany (1991) Proc. Natl. Acad. Sci USA 88:189). In suchcases, ligation will occur only if there is a perfect match at the 3′end of the 5′ sequence making it possible to detect the presence of aknown mutation at a specific site by looking for the presence or absenceof amplification.

In another aspect, the invention features a set of oligonucleotides. Theset includes a plurality of oligonucleotides, each of which is at leastpartially complementary (e.g., at least 50%, 60%, 70%, 80%, 90%, 92%,95%, 97%, 98%, or 99% complementary) to a 52906, 33408, or 12189 nucleicacid.

In a preferred embodiment the set includes a first and a secondoligonucleotide. The first and second oligonucleotide can hybridize tothe same or to different locations of SEQ ID NO:1, SEQ ID NO:4, or SEQID NO:7 or the complement of SEQ ID NO:1, SEQ ID NO:4, or SEQ ID NO:7.Different locations can be different but overlapping, or non-overlappingon the same strand. The first and second oligonucleotide can hybridizeto sites on the same or on different strands.

The set can be useful, e.g., for identifying SNP's, or identifyingspecific alleles of 52906, 33408, or 12189. In a preferred embodiment,each oligonucleotide of the set has a different nucleotide at aninterrogation position. In one embodiment, the set. includes twooligonucleotides, each complementary to a different allele at a locus,e.g., a biallelic or polymorphic locus.

In another embodiment, the set includes four oligonucleotides, eachhaving a different nucleotide (e.g., adenine, guanine, cytosine, orthymidine) at the interrogation position. The interrogation position canbe a SNP or the site of a mutation. In another preferred embodiment, theoligonucleotides of the plurality are identical in sequence to oneanother (except for differences in length). The oligonucleotides can beprovided with differential labels, such that an oligonucleotide thathybridizes to one allele provides a signal that is distinguishable froman oligonucleotide that hybridizes to a second allele. In still anotherembodiment, at least one of the oligonucleotides of the set has anucleotide change at a position in addition to a query position, e.g., adestabilizing mutation to decrease the T_(m) of the oligonucleotide. Inanother embodiment, at least one oligonucleotide of the set has anon-natural nucleotide, e.g., inosine. In a preferred embodiment, theoligonucleotides are attached to a solid support, e.g., to differentaddresses of an array or to different beads or nanoparticles.

In a preferred embodiment the set of oligo nucleotides can be used tospecifically amplify, e.g., by PCR, or detect, a 52906, 33408, or 12189nucleic acid.

The methods described herein may be performed, for example, by utilizingpre-packaged diagnostic kits comprising at least one probe nucleic acidor antibody reagent described herein, which may be conveniently used,e.g., in clinical settings to diagnose patients exhibiting symptoms orfamily history of a disease or illness involving a 52906, 33408, or12189 gene.

Use of 52906, 33408, or 12189 Molecules as Surrogate Markers

The 52906, 33408, or 12189 molecules of the invention are also useful asmarkers of disorders or disease states, as markers for precursors ofdisease states, as markers for predisposition of disease states, asmarkers of drug activity, or as markers of the pharmacogenomic profileof a subject. Using the methods described herein, the presence, absenceand/or quantity of the 52906, 33408, or 12189 molecules of the inventionmay be detected, and may be correlated with one or more biologicalstates in vivo. For example, the 52906, 33408, or 12189 molecules of theinvention may serve as surrogate markers for one or more disorders ordisease states or for conditions leading up to disease states. As usedherein, a “surrogate marker” is an objective biochemical marker whichcorrelates with the absence or presence of a disease or disorder, orwith the progression of a disease or disorder (e.g., with the presenceor absence of a tumor). The presence or quantity of such markers isindependent of the disease. Therefore, these markers may serve toindicate whether a particular course of treatment is effective inlessening a disease state or disorder. Surrogate markers are ofparticular use when the presence or extent of a disease state ordisorder is difficult to assess through standard methodologies (e.g.,early stage tumors), or when an assessment of disease progression isdesired before a potentially dangerous clinical endpoint is reached(e.g., an assessment of cardiovascular disease may be made usingcholesterol levels as a surrogate marker, and an analysis of HIVinfection may be made using HIV RNA levels as a surrogate marker, wellin advance of the undesirable clinical outcomes of myocardial infarctionor fully-developed AIDS). Examples of the use of surrogate markers inthe art include: Koomen et al (2000) J. Mass. Spectrom. 35: 258-264; andJames (1994) AIDS Treatment News Archive 209.

The 52906, 33408, or 12189 molecules of the invention are also useful aspharmacodynamic markers. As used herein, a “pharmacodynamic marker” isan objective biochemical marker which correlates specifically with drugeffects. The presence or quantity of a pharmacodynamic marker is notrelated to the disease state or disorder for which the drug is beingadministered; therefore, the presence or quantity of the marker isindicative of the presence or activity of the drug in a subject. Forexample, a pharmacodynamic marker may be indicative of the concentrationof the drug in a biological tissue, in that the marker is eitherexpressed or transcribed or not expressed or transcribed in that tissuein relationship to the level of the drug. In this fashion, thedistribution or uptake of the drug may be monitored by thepharmacodynamic marker. Similarly, the presence or quantity of thepharmacodynamic marker may be related to the presence or quantity of themetabolic product of a drug, such that the presence or quantity of themarker is indicative of the relative breakdown rate of the drug in vivo.Pharmacodynamic markers are of particular use in increasing thesensitivity of detection of drug effects, particularly when the drug isadministered in low doses. Since even a small amount of a drug may besufficient to activate multiple rounds of marker (e.g., a 52906, 33408,or 12189 marker) transcription or expression, the amplified marker maybe in a quantity which is more readily detectable than the drug itself.Also, the marker may be more easily detected due to the nature of themarker itself; for example, using the methods described herein,anti-52906, 33408, or 12189 antibodies may be employed in animmune-based detection system for a 52906, 33408, or 12189 proteinmarker, or 52906, 33408, or 12189-specific radiolabeled probes may beused to detect a 52906, 33408, or 12189 mRNA marker. Furthermore, theuse of a pharmacodynamic marker may offer mechanism-based prediction ofrisk due to drug treatment beyond the range of possible directobservations. Examples of the use of pharmacodynamic markers in the artinclude: Matsuda et al. U.S. Pat. No. 6,033,862; Hattis et al. (1991)Env. Health Perspect. 90: 229-238; Schentag (1999) Am. J. Health-Syst.Pharm. 56 Suppl. 3: S21-S24; and Nicolau (1999) Am, J. Health-Syst.Pharm. 56 Suppl. 3: S16-S20.

The 52906, 33408, or 12189 molecules of the invention are also useful aspharmacogenomic markers. As used herein, a “pharmacogenomic marker” isan objective biochemical marker which correlates with a specificclinical drug response or susceptibility in a subject (see, e.g., McLeodet al. (1999) Eur. J. Cancer 35: 1650-1652). The presence or quantity ofthe pharmacogenomic marker is related to the predicted response of thesubject to a specific drug or class of drugs prior to administration ofthe drug. By assessing the presence or quantity of one or morepharmacogenomic markers in a subject, a drug therapy which is mostappropriate for the subject, or which is predicted to have a greaterdegree of success, may be selected. For example, based on the presenceor quantity of RNA, or protein (e.g., 52906, 33408, or 12189 protein orRNA) for specific tumor markers in a subject, a drug or course oftreatment may be selected that is optimized for the treatment of thespecific tumor likely to be present in the subject. Similarly, thepresence or absence of a specific sequence mutation in 52906, 33408, or12189 DNA may correlate 52906, 33408, or 12189 drug response. The use ofpharmacogenomic markers therefore permits the application of the mostappropriate treatment for each subject without having to administer thetherapy.

Pharmaceutical Compositions

The nucleic acid and polypeptides, fragments thereof, as well asanti-52906, 33408, or 12189 antibodies (also referred to herein as“active compounds”) of the invention can be incorporated intopharmaceutical compositions. Such compositions typically include thenucleic acid molecule, protein, or antibody and a pharmaceuticallyacceptable carrier. As used herein the language “pharmaceuticallyacceptable carrier” includes solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like, compatible with pharmaceutical administration.Supplementary active compounds can also be incorporated into thecompositions.

A pharmaceutical composition is formulated to be compatible with itsintended route of administration. Examples of routes of administrationinclude parenteral, e.g., intravenous, intradermal, subcutaneous, oral(e.g., inhalation), transdermal (topical), transmucosal, and rectaladministration. Solutions or suspensions used for parenteral,intradermal, or subcutaneous application can include the followingcomponents: a sterile diluent such as water for injection, salinesolution, fixed oils, polyethylene glycols, glycerine, propylene glycolor other synthetic solvents; antibacterial agents such as benzyl alcoholor methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It should be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules, e.g., gelatin capsules. Oral compositionscan also be prepared using a fluid carrier for use as a mouthwash.Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is advantageous to formulate oral or parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the subject to be treated; each unitcontaining a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds which exhibit high therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC50 (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

As defined herein, a therapeutically effective amount of protein orpolypeptide (i.e., an effective dosage) ranges from about 0.001 to 30mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, morepreferably about 0.1 to 20 mg/kg body weight, and even more preferablyabout 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6mg/kg body weight. The protein or polypeptide can be administered onetime per week for between about 1 to 10 weeks, preferably between 2 to 8weeks, more preferably between about 3 to 7 weeks, and even morepreferably for about 4, 5, or 6 weeks. The skilled artisan willappreciate that certain factors may influence the dosage and timingrequired to effectively treat a subject, including but not limited tothe severity of the disease or disorder, previous treatments, thegeneral health and/or age of the subject, and other diseases present.Moreover, treatment of a subject with a therapeutically effective amountof a protein, polypeptide, or antibody can include a single treatmentor, preferably, can include a series of treatments.

For antibodies, the preferred dosage is 0.1 mg/kg of body weight(generally 10 mg/kg to 20 mg/kg). If the antibody is to act in thebrain, a dosage of 50 mg/kg to 100 mg/kg is usually appropriate.Generally, partially human antibodies and fully human antibodies have alonger half-life within the human body than other antibodies.Accordingly, lower dosages and less frequent administration is oftenpossible. Modifications such as lipidation can be used to stabilizeantibodies and to enhance uptake and tissue penetration (e.g., into thebrain). A method for lipidation of antibodies is described by Cruikshanket al. ((1997) J. Acquired Immune Deficiency Syndromes and HumanRetrovirology 14:193).

The present invention encompasses agents which modulate expression oractivity. An agent may, for example, be a small molecule. For example,such small molecules include, but are not limited to, peptides,peptidomimetics (e.g., peptoids), amino acids, amino acid analogs,polynucleotides, polynucleotide analogs, nucleotides, nucleotideanalogs, organic or inorganic compounds (i.e.,. including heteroorganicand organometallic compounds) having a molecular weight less than about10,000 grams per mole, organic or inorganic compounds having a molecularweight less than about 5,000 grams per mole, organic or inorganiccompounds having a molecular weight less than about 1,000 grams permole, organic or inorganic compounds having a molecular weight less thanabout 500 grams per mole, and salts, esters, and other pharmaceuticallyacceptable forms of such compounds.

Exemplary doses include milligram or microgram amounts of the smallmolecule per kilogram of subject or sample weight (e.g., about 1microgram per kilogram to about 500 milligrams per kilogram, about 100micrograms per kilogram to about 5 milligrams per kilogram, or about 1microgram per kilogram to about 50 micrograms per kilogram. It isfurthermore understood that appropriate doses of a small molecule dependupon the potency of the small molecule with respect to the expression oractivity to be modulated. When one or more of these small molecules isto be administered to an animal (e.g., a human) in order to modulateexpression or activity of a polypeptide or nucleic acid of theinvention, a physician, veterinarian, or researcher may, for example,prescribe a relatively low dose at first, subsequently increasing thedose until an appropriate response is obtained. In addition, it isunderstood that the specific dose level for any particular animalsubject will depend upon a variety of factors including the activity ofthe specific compound employed, the age, body weight, general health,gender, and diet of the subject, the time of administration, the routeof administration, the rate of excretion, any drug combination, and thedegree of expression or activity to be modulated.

An antibody (or fragment thereof) may be conjugated to a therapeuticmoiety such as a cytotoxin, a therapeutic agent or a radioactive metalion. A cytotoxin or cytotoxic agent includes any agent that isdetrimental to cells. Examples include taxol, cytochalasin B, gramicidinD, ethidium bromide, emetine, mitomycin, etoposide, tenoposide,vincristine, vinblastine, coichicin, doxorubicin, daunorubicin,dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D,1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine,propranolol, and puromycin and analogs or homologs thereof. Therapeuticagents include, but are not limited to, antimetabolites (e.g.,methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine,thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC, and cis-dichlorodiamine platinum (II) (DDP) cisplatin),anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

The conjugates of the invention can be used for modifying a givenbiological response, the drug moiety is not to be construed as limitedto classical chemical therapeutic agents. For example, the drug moietymay be a protein or polypeptide possessing a desired biologicalactivity. Such proteins may include, for example, a toxin such as abrin,ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such astumor necrosis factor, α-interferon, β-interferon, nerve growth factor,platelet derived growth factor, tissue plasminogen activator; or,biological response modifiers such as, for example, lymphokines,interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”),granulocyte macrophase colony stimulating factor (“GM-CSF”), granulocytecolony stimulating factor (“G-CSF”), or other growth factors.

Alternatively, an antibody can be conjugated to a second antibody toform an antibody heteroconjugate as described by Segal in U.S. Pat. No.4,676,980.

The nucleic acid molecules of the invention can be inserted into vectorsand used as gene therapy vectors. Gene therapy vectors can be deliveredto a subject by, for example, intravenous injection, localadministration (see U.S. Pat. No. 5,328,470) or by stereotacticinjection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA91:3054-3057). The.pharmaceutical preparation of the gene therapy vectorcan include the gene therapy vector in an acceptable diluent, or cancomprise a slow release matrix in which the gene delivery vehicle isimbedded. Alternatively, where the complete gene delivery vector can beproduced intact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can include one or more cells which producethe gene delivery system.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

Methods of Treatment

The present invention provides for both prophylactic and therapeuticmethods of treating a subject at risk of (or susceptible to) a disorderor having a disorder associated with aberrant or unwanted 52906, 33408,or 12189 expression or activity. As used herein, the term “treatment” isdefined as the application or administration of a therapeutic agent to apatient, or application or administration of a therapeutic agent to anisolated tissue or cell line from a patient, who has a disease, asymptom of disease or a predisposition toward a disease, with thepurpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate,improve or affect the disease, the symptoms of disease or thepredisposition toward disease. A therapeutic agent includes, but is notlimited to, small molecules, peptides, antibodies, ribozymes andantisense oligonucleotides.

With regards to both prophylactic and therapeutic methods of treatment,such treatments may be specifically tailored or modified, based onknowledge obtained from the field of pharmacogenomics.“Pharmacogenomics”, as used herein, refers to the application ofgenomics technologies such as gene sequencing, statistical genetics, andgene expression analysis to drugs in clinical development and on themarket. More specifically, the term refers the study of how a patient'sgenes determine his or her response to a drug (e.g., a patient's “drugresponse phenotype”, or “drug:response genotype”.) Thus, another aspectof the invention provides methods for tailoring an individual'sprophylactic or therapeutic treatment with either the 52906, 33408, or12189 molecules of the present invention or 52906, 33408, or 12189modulators according to that individual's drug response genotype.Pharmacogenomics allows a clinician or physician to target prophylacticor therapeutic treatments to patients who will most benefit from thetreatment and to avoid treatment of patients who will experience toxicdrug-related side effects.

In one aspect, the invention provides a method for preventing in asubject, a disease or condition associated with an aberrant or unwanted52906, 33408, or 12189 expression or activity, by administering to thesubject a 52906, 33408, or 12189 or an agent which modulates 52906,33408, or 12189 expression or at least one 52906, 33408, or 12189activity. Subjects at risk for a disease which is caused or contributedto by aberrant or unwanted 52906, 33408, or 12189 expression or activitycan be identified by, for example, any or a combination of diagnostic orprognostic assays as described herein. Administration of a prophylacticagent can occur prior to the manifestation of symptoms characteristic ofthe 52906, 33408, or 12189 aberrance, such that a disease or disorder isprevented or, alternatively, delayed in its progression. Depending onthe type of 52906, 33408, or 12189 aberrance, for example, a 52906,33408, or 12189, 52906, 33408, or 12189 agonist or 52906, 33408, or12189 antagonist agent can be used for treating the subject. Theappropriate agent can be determined based on screening assays describedherein.

It is possible that some 52906, 33408, or 12189 disorders can be caused,at least in part, by an abnormal level of gene product, or by thepresence of a gene product exhibiting abnormal activity. As such, thereduction in the level and/or activity of such gene products would bringabout the amelioration of disorder symptoms.

The 52906, 33408, or 12189 molecules can act as novel diagnostic targetsand therapeutic agents for controlling one or more of cellularproliferative and/or differentiative disorders, disorders associatedwith bone metabolism, immune disorders, liver disorders, viral diseases,pain or metabolic disorders.

Examples of cellular proliferative and/or differentiative disordersinclude cancer, e.g., carcinoma, sarcoma, metastatic disorders orhematopoietic neoplastic disorders, e.g., leukemias. A metastatic tumorcan arise from a multitude of primary tumor types, including but notlimited to those of prostate, colon, lung, breast and liver origin.

As used herein, the terms “cancer”, “hyperproliferative” and“neoplastic” refer to cells having the capacity for autonomous growth,i.e., an abnormal state or condition characterized by rapidlyproliferating cell growth. Hyperproliferative and neoplastic diseasestates may be categorized as pathologic, i.e., characterizing orconstituting a disease state, or may be categorized as non-pathologic,i.e., a deviation from normal but not associated with a disease state.The term is meant to include all types of cancerous growths or oncogenicprocesses, metastatic tissues or malignantly transformed cells, tissues,or organs, irrespective of histopathologic type or stage ofinvasiveness. “Pathologic hyperproliferative” cells occur in diseasestates characterized by malignant tumor growth. Examples ofnon-pathologic hyperproliferative cells include proliferation of cellsassociated with wound repair.

The terms “cancer” or “neoplasms” include malignancies of the variousorgan systems, such as affecting lung, breast, thyroid, lymphoid,gastrointestinal, and genito-urinary tract, as well as adenocarcinomaswhich include malignancies such as most colon cancers, renal-cellcarcinoma, prostate cancer and/or testicular tumors, non-small cellcarcinoma of the lung, cancer of the small intestine and cancer of theesophagus.

The term “carcinoma” is art recognized and refers to malignancies ofepithelial or endocrine tissues including respiratory system carcinomas,gastrointestinal system carcinomas, genitourinary system carcinomas,testicular carcinomas, breast carcinomas, prostatic carcinomas,endocrine system carcinomas, and melanomas. Exemplary carcinomas includethose forming from tissue of the cervix, lung, prostate, breast, headand neck, colon and ovary. The term also includes carcinosarcomas, e.g.,which include malignant tumors composed of carcinomatous and sarcomatoustissues. An “adenocarcinoma” refers to a.carcinoma derived fromglandular tissue or in which the tumor cells form recognizable glandularstructures.

The term “sarcoma” is art recognized and refers to malignant tumors ofmesenchymal derivation.

Additional examples of proliferative disorders include hematopoieticneoplastic disorders. As used herein, the term “hematopoietic neoplasticdisorders” includes diseases involving hyperplastic/neoplastic cells ofhematopoietic origin, e.g., arising from myeloid, lymphoid or erythroidlineages, or precursor cells thereof. Preferably, the diseases arisefrom poorly differentiated acute leukemias, e.g., erythroblasticleukemia and acute megakaryoblastic leukemia. Additional exemplarymyeloid disorders include, but are not limited to, acute promyeloidleukemia (APML), acute myelogenous leukemia (AML) and chronicmyelogenous leukemia (CML) (reviewed in Vaickus, L. (1991) Crit Rev. inOncol./Hemotol. 11:267-97); lymphoid malignancies include, but are notlimited to acute lymphoblastic leukemia (ALL) which includes B-lineageALL and T-lineage ALL, chronic lymphocytic leukemia (CLL),prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) andWaldenstrom's macroglobulinemia (WM). Additional forms of malignantlymphomas include, but are not limited to non-Hodgkin lymphoma andvariants thereof, peripheral T cell lymphomas, adult T cellleukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), largegranular lymphocytic leukemia (LGF), Hodgkin's disease andReed-Sternberg disease.

Aberrant. expression and/or activity of 52906, 33408, or 12189 moleculesmay mediate disorders associated with bone metabolism. “Bone metabolism”refers to direct or indirect effects in the formation or degeneration ofbone structures, e.g., bone formation, bone resorption, etc., which mayultimately affect the concentrations in serum of calcium and phosphate.This term also includes activities mediated by 52906, 33408, or 12189molecules effects in bone cells, e.g. osteoclasts and osteoblasts, thatmay in turn result in bone formation and degeneration. For example,52906, 33408, or 12189 molecules may support different activities ofbone resorbing osteoclasts such as the stimulation of differentiation ofmonocytes and mononuclear phagocytes into osteoclasts. Accordingly,52906, 33408, or 12189 molecules that modulate the production of bonecells can influence bone formation and degeneration, and thus may beused to treat bone disorders. Examples of such disorders include, butare not limited to, osteoporosis, osteodystrophy, osteomalacia, rickets,osteitis fibrosa cystica, renal osteodystrophy, osteosclerosis,anti-convulsant treatment, osteopenia, fibrogenesis-imperfecta ossium,secondary hyperparathyrodism, hypoparathyroidism, hyperparathyroidism,cirrhosis, obstructive jaundice, drug induced metabolism, medullarycarcinoma, chronic renal disease, rickets, sarcoidosis, glucocorticoidantagonism, malabsorption syndrome, steatorrhea, tropical sprue,idiopathic hypercalcemia and milk fever.

The 52906, 33408, or 12189 nucleic acid and protein of the invention canbe used to treat and/or diagnose a variety of immune disorders. Examplesof immune disorders or diseases include, but are not limited to,autoimmune diseases (including, for example, diabetes mellitus,arthritis (including rheumatoid arthritis, juvenile rheumatoidarthritis, osteoarthritis, psoriatic arthritis), multiple sclerosis,encephalomyelitis, myasthenia gravis, systemic lupus erythematosis,autoimmune thyroiditis, dermatitis (including atopic dermatitis andeczematous dermatitis), psoriasis, Sjögren's Syndrome, Crohn's disease,aphthous ulcer, iritis, conjunctivitis, keratoconjunctivitis, ulcerativecolitis, asthma, allergic asthma, cutaneous lupus erythematosus,scleroderma, vaginitis, proctitis, drug eruptions, leprosy reversalreactions, erythema nodosum leprosum, autoimmune uveitis, allergicencephalomyelitis, acute necrotizing hemorrhagic encephalopathy,idiopathic bilateral progressive sensorineural hearing loss, aplasticanemia, pure red cell anemia, idiopathic thrombocytopenia,polychondritis, Wegener's granulomatosis, chronic active hepatitis,Stevens-Johnson syndrome, idiopathic sprue, lichen planus, Graves'disease, sarcoidosis, primary biliary cirrhosis, uveitis posterior, andinterstitial lung fibrosis), graft-versus-host disease, cases oftransplantation, and allergy such as, atopic allergy.

Disorders which may be treated or diagnosed by methods described hereininclude, but are not limited to, disorders associated with anaccumulation in the liver of fibrous tissue, such as that resulting froman imbalance between production and degradation of the extracellularmatrix accompanied by the collapse and condensation of preexistingfibers. The methods described herein can be used to diagnose or treathepatocellular necrosis or injury induced by a wide variety of agentsincluding processes which disturb homeostasis, such as an inflammatoryprocess, tissue damage resulting from toxic injury or altered hepaticblood flow, and infections (e.g., bacterial, viral and parasitic). Forexample, the methods can be used for the early detection of hepaticinjury, such as portal hypertension or hepatic fibrosis. In addition,the methods can be employed to detect liver fibrosis attributed toinborn errors of metabolism, for example, fibrosis resulting from astorage disorder such as Gaucher's disease (lipid abnormalities) or aglycogen storage disease, A1-antitrypsin deficiency; a disordermediating the accumulation (e.g., storage) of an exogenous substance,for example, hemochromatosis (iron-overload syndrome) and copper storagediseases (Wilson's disease), disorders resulting in the accumulation ofa toxic metabolite (e.g., tyrosinemia, fructosemia and galactosemia) andperoxisomal disorders (e.g., Zellweger syndrome). Additionally, themethods described herein may be useful for the early detection andtreatment of liver injury associated with the administration of variouschemicals or drugs, such as for example, methotrexate, isonizaid,oxyphenisatin, methyldopa, chlorpromazine, tolbutamide or alcohol, orwhich represents a hepatic manifestation of a vascular disorder such asobstruction of either the intrahepatic or extrahepatic bile flow or analteration in hepatic circulation resulting, for example, from chronicheart failure, veno-occlusive disease, portal vein thrombosis orBudd-Chiari syndrome.

Additionally, 52906, 33408, or 12189 molecules may play an importantrole in the etiology of certain viral diseases, including but notlimited to Hepatitis B, Hepatitis C and Herpes Simplex Virus (HSV).Modulators of 52906, 33408, or 12189 activity could be used to controlviral diseases. The modulators can be used in the treatment and/ordiagnosis of viral infected tissue or virus-associated tissue fibrosis,especially liver and liver fibrosis. Also, 52906, 33408, or 12189modulators can be used in the treatment and/or diagnosis ofvirus-associated carcinoma, especially hepatocellular cancer.

Additionally, 52906, 33408, or 12189 may play an important role in theregulation of metabolism or pain disorders. Diseases of metabolicimbalance include, but are not limited to, obesity, anorexia nervosa,cachexia, lipid disorders, and diabetes. Examples of pain disordersinclude, but. are not limited to, pain response elicited during variousforms of tissue injury, e.g., inflammation, infection, and ischemia,usually referred to as hyperalgesia (described in, for example, Fields,H. L. (1987) Pain, New York:McGraw-Hill); pain associated withmusculoskeletal disorders, e.g., joint pain;

tooth pain; headaches; pain associated with surgery; pain related toirritable bowel syndrome; or chest pain.

As discussed, successful treatment of 52906, 33408, or 12189 disorderscan be brought about by techniques that serve to inhibit the expressionor activity of target gene products. For example, compounds, e.g., anagent identified using an assays described above, that proves to exhibitnegative modulatory activity, can be used in accordance with theinvention to prevent and/or ameliorate symptoms of 52906, 33408, or12189 disorders. Such molecules can include, but are not limited topeptides, phosphopeptides, small organic or inorganic molecules, orantibodies (including, for example, polyclonal, monoclonal, humanized,anti-idiotypic, chimeric or single chain antibodies, and Fab, F(ab′)₂and Fab expression library fragments, scFV molecules, andepitope-binding fragments thereof).

Further, antisense and ribozyme molecules that inhibit expression of thetarget gene can also be used in accordance with the invention to reducethe level of target gene expression, thus effectively reducing the levelof target gene activity. Still firther, triple helix molecules can beutilized in reducing the level of target gene activity. Antisense,ribozyme and triple helix molecules are discussed above.

It is possible that the use of antisense, ribozyme, and/or triple helixmolecules to reduce or inhibit mutant gene expression can also reduce orinhibit the transcription (triple helix) and/or translation (antisense,ribozyme) of mRNA produced by normal target gene alleles, such that theconcentration of normal target gene product present can be lower than isnecessary for a normal phenotype. In such cases, nucleic acid moleculesthat encode and express target gene polypeptides exhibiting normaltarget gene activity can be introduced into cells via gene therapymethod. Alternatively, in instances in that the target gene encodes anextracellular protein, it can be preferable to co-administer normaltarget gene protein into the cell or tissue in order to maintain therequisite level of cellular or tissue target gene activity.

Another method by which nucleic acid molecules may be utilized intreating or preventing a disease characterized by 52906, 33408, or 12189expression is through the use of aptamer molecules specific for 52906,33408, or 12189 protein. Aptamers are nucleic acid molecules having atertiary structure which permits them to specifically bind to proteinligands (see, e.g., Osborne, et al. (1997) Curr. Opin. Chem Biol. 1:5-9; and Patel, D. J. (1997) Curr Opin Chem Biol 1:32-46). Since nucleicacid molecules may in many cases be more conveniently introduced intotarget cells than therapeutic protein molecules may be, aptamers offer amethod by which 52906, 33408, or 12189 protein activity may bespecifically decreased without the introduction of drugs or othermolecules which may have pluripotent effects.

Antibodies can be generated that are both specific for target geneproduct and that reduce target gene product activity. Such antibodiesmay, therefore, by administered in instances whereby negative modulatorytechniques are appropriate for the treatment of 52906, 33408, or 12189disorders. For a description of antibodies, see the Antibody sectionabove.

In circumstances wherein injection of an animal or a human subject witha 52906, 33408, or 12189 protein or epitope for stimulating antibodyproduction is harmful to the subject, it is possible to generate animmune response against 52906, 33408, or 12189 through the use ofanti-idiotypic antibodies (see, for example, Herlyn, D. (1999) Ann Med31:66-78; and Bhattacharya-Chatterjee, M., and Foon, K. A. (1998) CancerTreat Res. 94:51-68). If an anti-idiotypic antibody is introduced into amammal or human subject, it should stimulate the production ofanti-anti-idiotypic antibodies, which should be specific to the 52906,33408, or 12189 protein. Vaccines directed to a disease characterized by52906, 33408, or 12189 expression may also be generated in this fashion.

In instances where the target antigen is intracellular and wholeantibodies are used, internalizing antibodies may be preferred.Lipofectin or liposomes can be used to deliver the antibody or afragment of the Fab region that binds to the target antigen into cells.Where fragments of the antibody are used, the smallest inhibitoryfragment that binds to the target antigen is preferred. For example,peptides having an amino acid sequence corresponding to the Fv region ofthe antibody can be used. Alternatively, single chain neutralizingantibodies that bind to intracellular target antigens can also beadministered. Such single chain antibodies can be administered, forexample, by expressing nucleotide sequences encoding single-chainantibodies within the target cell population (see e.g., Marasco et al.(1993) Proc. Natl. Acad. Sci. USA 90:7889-7893).

The identified compounds that inhibit target gene expression, synthesisand/or activity can be administered to a patient at therapeuticallyeffective doses to prevent, treat or ameliorate 52906, 33408, or 12189disorders. A therapeutically effective dose refers to that amount of thecompound sufficient to result in amelioration of symptoms of thedisorders. Toxicity and therapeutic efficacy of such compounds can bedetermined by standard pharmaceutical procedures as described above.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage can vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose can beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound that achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma can bemeasured, for example, by high performance liquid chromatography.

Another example of determination of effective dose for an individual isthe ability to directly assay levels of “free” and “bound” compound inthe serum of the test subject. Such assays may utilize antibody mimicsand/or “biosensors” that have been created through molecular imprintingtechniques. The compound which is able to modulate 52906, 33408, or12189 activity is used as a template, or “imprinting molecule”, tospatially organize polymerizable monomers prior to their polymerizationwith catalytic reagents. The subsequent removal of the imprintedmolecule leaves a polymer matrix which contains a repeated. “negativeimage” of the compound and is able to selectively rebind the moleculeunder biological assay conditions. A detailed review of this techniquecan be seen in Ansell, R. J. et al (1996) Current Opinion inBiotechnology 7:89-94 and in Shea, K. J. (1994) Trends in PolymerScience 2:166-173. Such “imprinted” affinity matrixes are amenable toligand-binding assays, whereby the immobilized monoclonal antibodycomponent is replaced by an appropriately imprinted matrix. An exampleof the use of such matrixes in this way can be seen in Vlatakis, G. etal (1993) Nature 361:645-647. Through the use of isotope-labeling, the“free” concentration of compound which modulates the expression oractivity of 52906, 33408, or 12189 can be readily monitored and used incalculations of IC₅₀.

Such “imprinted” affinity matrixes can also be designed to includefluorescent groups whose photon-emitting properties measurably changeupon local and selective binding of target compound. These changes canbe readily assayed in real time using appropriate fiberoptic devices, inturn allowing the dose in a test subject to be quickly optimized basedon its individual IC₅₀. An rudimentary example of such a “biosensor” isdiscussed in Kriz, D. et al (1995) Analytical Chemistry 67:2142-2144.

Another aspect of the invention pertains to methods of modulating 52906,33408, or 12189 expression or activity for therapeutic purposes.Accordingly, in an exemplary embodiment, the modulatory method of theinvention involves contacting a cell with a 52906, 33408, or 12189 oragent that modulates one or more of the activities of 52906, 33408, or12189 protein activity associated with the cell. An agent that modulates52906, 33408, or 12189 protein activity can be an agent as describedherein, such as a nucleic acid or a protein, a naturally-occurringtarget molecule of a 52906, 33408, or 12189 protein (e.g., a 52906,33408, or 12189 substrate or receptor), a 52906, 33408, or 12189antibody, a 52906, 33408, or 12189 agonist or antagonist, apeptidomimetic of a 52906, 33408, or 1-2189 agonist or antagonist, orother small molecule.

In one embodiment, the agent stimulates one or 52906, 33408, or 12189activities. Examples of such stimulatory agents include active 52906,33408, or 12189 protein and a nucleic acid molecule encoding 52906,33408, or 12189. In another embodiment, the agent inhibits one or more52906, 33408, or 12189 activities. Examples of such inhibitory agentsinclude antisense 52906, 33408, or 12189 nucleic acid molecules,anti-52906, 33408, or 12189 antibodies, and 52906, 33408, or 12189inhibitors. These modulatory methods can be performed in vitro (e.g., byculturing the cell with the agent) or, alternatively, in vivo (e.g., byadministering the agent to a subject). As such, the present inventionprovides methods of treating an individual afflicted with a disease ordisorder characterized by aberrant or unwanted expression or activity ofa 52906, 33408, or 12189 protein or nucleic acid molecule. In oneembodiment, the method involves administering an agent (e.g., an agentidentified by a screening assay described herein), or combination ofagents that modulates (e.g., up regulates or down regulates) 52906,33408, or 12189 expression or activity. In another embodiment, themethod involves administering a 52906, 33408, or 12189 protein ornucleic acid molecule as therapy to compensate for reduced, aberrant, orunwanted 52906, 33408, or 12189 expression or activity.

Stimulation of 52906, 33408, or 12189 activity is desirable insituations in which 52906, 33408, or 12189 is abnormally downregulatedand/or in which increased 52906, 33408, or 12189 activity is likely tohave a beneficial effect. For example, stimulation of 52906, 33408, or12189 activity is desirable in situations in which a 52906, 33408, or12189 is downregulated and/or in which increased 52906, 33408, or 12189activity is likely to have a beneficial effect. Likewise, inhibition of52906, 33408, or 12189 activity is desirable in situations in which52906, 33408, or 12189 is abnormally upregulated and/or in whichdecreased 52906, 33408, or 12189 activity is likely to have a beneficialeffect.

Pharmacogenomics

The 52906, 33408, or 12189 molecules of the present invention, as wellas agents, or modulators which have a stimulatory or inhibitory effecton 52906, 33408, or 12189 activity (e.g., 52906, 33408, or 12189 geneexpression) as identified by a screening assay described herein can beadministered to individuals to treat (prophylactically ortherapeutically) 52906, 33408, or 12189 associated disorders (e.g., adisorder characterized by abnormal ion flux such as a neurologicaldisorder or a cardiac disorder) associated with aberrant or unwanted52906, 33408, or 12189 activity. In conjunction with such treatment,pharmacogenomics (i.e., the study of the relationship between anindividual's genotype and that individual's response to a foreigncompound or drug) may be considered. Differences in metabolism oftherapeutics can lead to severe toxicity or therapeutic failure byaltering the relation between dose and blood concentration of thepharmacologically active drug. Thus, a physician or clinician mayconsider applying knowledge obtained in relevant pharmacogenomicsstudies in determining whether to administer a 52906, 33408, or 12189molecule or 52906, 33408, or 12189 modulator as well as tailoring thedosage and/or therapeutic regimen of treatment with a 52906, 33408,. or12189 molecule or 52906, 33408, or 12189 modulator.

Pharmacogenomics deals with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. See, for example, Eichelbaum, M. et al.(1996) Clin. Exp. Pharmacol. Physiol. 23:983-985 and Linder, M. W. etal. (1997) Clin. Chem. 43:254-266. In general, two types ofpharmacogenetic conditions can be differentiated. Genetic conditionstransmitted as a single factor altering the way drugs act on the body(altered drug action) or genetic conditions transmitted as singlefactors altering the way the body acts on drugs (altered drugmetabolism). These pharmacogenetic conditions can occur either as raregenetic defects or as naturally-occurring polymorphisms. For example,glucose-6-phosphate dehydrogenase deficiency (G6PD) is a commoninherited enzymopathy in which the main clinical complication ishaemolysis after ingestion of oxidant drugs (anti-malarials,sulfonamides, analgesics, nitrofurans) and consumption of fava beans.

One pharmacogenomics approach to identifying genes that predict drugresponse, known as “a genome-wide association”, relies primarily on ahigh-resolution map of the human genome consisting of already knowngene-related markers (e.g., a “bi-allelic” gene marker map whichconsists of 60,000-100,000 polymorphic or variable sites on the humangenome, each of which has two variants.) Such a high-resolution geneticmap can be compared to a map of the genome of each of a statisticallysignificant number of patients taking part in a Phase II/III drug trialto identify markers associated with a particular observed drug responseor side effect. Alternatively, such a high resolution map can begenerated from a combination of some ten-million known single nucleotidepolymorphisms (SNPs) in the human genome. As used herein, a “SNP” is acommon alteration that occurs in a single nucleotide base in a stretchof DNA. For example, a SNP may occur once per every 1000 bases of DNA. ASNP may be involved in a disease process, however, the vast majority maynot be disease-associated. Given a genetic map based on the occurrenceof such SNPs, individuals can be grouped into genetic categoriesdepending on a particular pattern of SNPs in their individual genome. Insuch a manner, treatment regimens can be tailored to groups ofgenetically similar individuals, taking into account traits that may becommon among such genetically similar individuals.

Alternatively, a method termed the “candidate gene approach,” can beutilized to identify genes that predict drug response. According to thismethod, if a gene that encodes a drug's target is known (e.g., a 52906,33408, or 12189 protein of the present invention), all common variantsof that gene can be fairly easily identified in the population and itcan be determined if having one version of the gene versus another isassociated with a particular drug response.

Alternatively, a method termed the “gene expression profiling,” can beutilized to identify genes that predict drug response. For example, thegene expression of an animal dosed with a drug (e.g., a 52906, 33408, or12189 molecule or 52906, 33408, or 12189 modulator of the presentinvention) can give an indication whether gene pathways related totoxicity have been turned on.

Information generated from more than one of the above pharmacogenomicsapproaches can be used to determine appropriate dosage and treatmentregimens for prophylactic or therapeutic treatment of an individual.This knowledge, when applied to dosing or drug selection, can avoidadverse reactions or therapeutic failure and thus enhance therapeutic orprophylactic efficiency when treating a subject with a 52906, 33408, or12189 molecule or 52906, 33408, or 12189 modulator, such as a modulatoridentified by one of the exemplary screening assays described herein.

The present invention further provides methods for identifying newagents, or combinations, that are based on identifying agents thatmodulate the activity of one or more of the gene products encoded by oneor more of the 52906, 33408, or 12189 genes of the present invention,wherein these products may be associated with resistance of the cells toa therapeutic agent. Specifically, the activity of the proteins encodedby the 52906, 33408, or 12189 genes of the present invention can be usedas a basis for identifying agents for overcoming agent resistance. Byblocking the activity of one or more of the resistance proteins, targetcells, e.g., human cells, will become sensitive to treatment with anagent that the unmodified target cells were resistant to.

Monitoring the influence of agents (e.g., drugs) on the expression oractivity of a 52906, 33408, or 12189 protein can be applied in clinicaltrials. For example, the effectiveness of an agent determined by ascreening assay as described herein to increase 52906, 33408, or 12189gene expression, protein levels, or upregulate 52906, 33408, or 12189activity, can be monitored in clinical trials of subjects exhibitingdecreased 52906, 33408, or 12189 gene expression, protein levels, ordownregulated 52906, 33408, or 12189 activity. Alternatively, theeffectiveness of an agent determined by a screening assay to decrease52906, 33408, or 12189 gene expression, protein levels, or downregulate52906, 33408, or 12189 activity, can be monitored in clinical trials ofsubjects exhibiting increased 52906, 33408, or 12189 gene expression,protein levels, or upregulated 52906, 33408, or 12189 activity. In suchclinical trials, the expression or activity of a 52906, 33408, or 12189gene, and preferably, other genes that have been implicated in, forexample, a 52906, 33408, or 12189-associated disorder can be used as a“read out” or markers of the phenotype of a particular cell.

52906, 33408, or 12189 Informatics

The sequence of a 52906, 33408, or 12189 molecule is provided in avariety of media to facilitate use thereof. A sequence can be providedas a manufacture, other than an isolated nucleic acid or amino acidmolecule, which contains a 52906, 33408, or 12189. Such a manufacturecan provide a nucleotide or amino acid sequence, e.g., an open readingframe, in a form which allows examination of the manufacture using meansnot directly applicable to examining the nucleotide or amino acidsequences, or a subset thereof, as they exists in nature or in purifiedform. The sequence information can include, but is not limited to,52906, 33408, or 12189 full-length nucleotide and/or amino acidsequences, partial nucleotide and/or amino acid sequences, polymorphicsequences including single nucleotide polymorphisms (SNPs), epitopesequence, and the like. In a preferred embodiment, the manufacture is amachine-readable medium, e.g., a magnetic, optical, chemical ormechanical information storage device.

As used herein, “machine-readable media” refers to any medium that canbe read and accessed directly by a machine, e.g., a digital computer oranalogue computer. Non-limiting examples of a computer include a desktopPC, laptop, mainframe, server (e.g., a web server, network server, orserver farm), handheld digital assistant, pager, mobile telephone, andthe like. The computer can be stand-alone or connected to acommunications network, e.g., a local area network (such as a VPN orintranet), a wide area network (e.g., an Extranet or the Internet), or atelephone network (e.g., a wireless, DSL, or ISDN network).Machine-readable media include, but are not limited to: magnetic storagemedia, such as floppy discs, hard disc storage medium, and magnetictape; optical storage media such as CD-ROM; electrical storage mediasuch as RAM, ROM, EPROM, EEPROM, flash memory, and the like; and hybridsof these categories such as magnetic/optical storage media.

A variety of data storage structures are available to a skilled artisanfor creating a machine-readable medium having recorded thereon anucleotide or amino acid sequence of the present invention. The choiceof the data storage structure will generally be based on the meanschosen to access the stored information. In addition, a variety of dataprocessor programs and formats can be used to store the nucleotidesequence information of the present invention on computer readablemedium. The sequence information can be represented in a word processingtext file, formatted in commercially-available software such asWordPerfect and Microsoft Word, or represented in the form of an ASCIIfile, stored in a database application, such as DB2, Sybase, Oracle, orthe like. The skilled artisan can readily adapt any number of dataprocessor structuring formats (e.g., text file or database) in order toobtain computer readable medium having recorded thereon the nucleotidesequence information of the present invention.

In a preferred embodiment, the sequence information is stored in arelational database (such as Sybase or Oracle). The database can have afirst table for storing sequence (nucleic acid and/or amino acidsequence) information. The sequence information can be stored in onefield (e.g., a first column) of a table row and an identifier for thesequence can be store in another field (e.g., a second column) of thetable row. The database can have a second table, e.g., storingannotations. The second table can have a field for the sequenceidentifier, a field for a descriptor or annotation text (e.g., thedescriptor can refer to a functionality of the sequence, a field for theinitial position in the sequence to which the annotation refers, and afield for the ultimate position in the sequence to which the annotationrefers. Non-limiting examples for annotation to nucleic acid sequencesinclude polymorphisms (e.g., SNP's) translational regulatory sites andsplice junctions. Non-limiting examples for annotations to amino acidsequence include polypeptide domains, e.g., a domain described herein;active sites and other functional amino acids; and modification sites.

By providing the nucleotide or amino acid sequences of the invention incomputer readable form, the skilled artisan can routinely access thesequence information for a variety of purposes. For example, one skilledin the art can use the nucleotide or amino acid sequences of theinvention in computer readable form to compare a target sequence ortarget structural motif with the sequence information stored within thedata storage means. A search is used to identify fragments or regions ofthe sequences of the invention which match a particular target sequenceor target motif. The search can be a BLAST search or other routinesequence comparison, e.g., a search described herein.

Thus, in one aspect, the invention features a method of analyzing 52906,33408, or 12189, e.g., analyzing structure, function, or relatedness toone or more other nucleic acid or amino acid sequences. The methodincludes: providing a 52906, 33408, or 12189 nucleic acid or amino acidsequence; comparing the 52906, 33408, or 12189 sequence with a secondsequence, e.g., one or more preferably a plurality of sequences from acollection of sequences, e.g., a nucleic acid or protein sequencedatabase to thereby analyze 52906, 33408, or 12189. The method can beperformed in a machine, e.g., a computer, or manually by a skilledartisan.

The method can include evaluating the sequence identity between a 52906,33408, or 12189 sequence and a database sequence. The method can beperformed by accessing the database at a second site, e.g., over theInternet.

As used herein, a “target sequence” can be any DNA or amino acidsequence of six or more nucleotides or two or more amino acids. Askilled artisan can readily recognize that the longer a target sequenceis, the less likely a target sequence will be present as a randomoccurrence in the database. Typical sequence lengths of a targetsequence are from about 10 to 100 amino acids or from about 30 to 300nucleotide residues. However, it is well recognized that commerciallyimportant fragments, such as sequence fragments involved in geneexpression and protein processing, may be of shorter length.

Computer software is publicly available which allows a skilled artisanto access sequence information provided in a computer readable mediumfor analysis and comparison to other sequences. A variety of knownalgorithms are disclosed publicly and a variety of commerciallyavailable software for conducting search means are and can be used inthe computer-based systems of the present invention. Examples of suchsoftware include, but are not limited to, MacPattern (EMBL), BLASTN andBLASTX (NCBI).

Thus, the invention features a method of making a computer readablerecord of a sequence of a 52906, 33408, or 12189 sequence which includesrecording the sequence on a computer readable matrix. In a preferredembodiment the record includes one or more of the following:identification of an ORF; identification of a domain, region, or site;identification of the start of transcription; identification of thetranscription terminator; the full length amino acid sequence of theprotein, or a mature form thereof; the 5′ end of the translated region.

In another aspect, the invention features, a method of analyzing asequence. The method includes: providing a 52906, 33408, or 12189sequence, or record, in machine-readable form; comparing a secondsequence to the 52906, 33408, or 12189 sequence; thereby analyzing asequence. Comparison can include comparing to sequences for sequenceidentity or determining if one sequence is included within the other,e.g., determining if the 52906, 33408, or 12189 sequence includes asequence being compared. In a preferred embodiment the 52906, 33408, or12189 or second sequence is stored on a first computer, e.g., at a firstsite and the comparison is performed, read, or recorded on a secondcomputer, e.g., at a second site. E.g., the 52906, 33408, or 12189 orsecond sequence can be stored in a public or proprietary database in onecomputer, and the results of the comparison performed, read, or recordedon a second computer. In a preferred embodiment the record includes oneor more of the following: identification of an ORF; identification of adomain, region, or site; identification of the start of transcription;identification of the transcription terminator; the full length aminoacid sequence of the protein, or a mature form thereof; the 5′ end ofthe translated region.

In another aspect, the invention provides a machine-readable medium forholding instructions for performing a method for determining whether asubject has a 52906, 33408, or 12189-associated disease or disorder or apre-disposition to a 52906, 33408, or 12189-associated disease ordisorder, wherein the method comprises the steps of determining 52906,33408, or 12189 sequence information associated with the subject andbased on the 52906, 33408, or 12189 sequence information, determiningwhether the subject has a 52906, 33408, or 12189-associated disease ordisorder or a pre-disposition to a 52906, 33408, or 12189-associateddisease or disorder and/or recommending a particular treatment for thedisease, disorder or pre-disease condition.

The invention further provides in an electronic system and/or in anetwork, a method for determining whether a subject has a 52906, 33408,or 12189-associated disease or disorder or a pre-disposition to adisease associated with a 52906, 33408, or 12189 wherein the methodcomprises the steps of determining 52906, 33408, or 12189 sequenceinformation associated with the subject, and based on the 52906, 33408,or 12189 sequence information, determining whether the subject has a52906, 33408, or 12189-associated disease or disorder or apre-disposition to a 52906, 33408, or 12189-associated disease ordisorder, and/or recommending a particular treatment for the disease,disorder or pre-disease condition. In a preferred embodiment, the methodfurther includes the step of receiving information, e.g., phenotypic orgenotypic information, associated with the subject and/or acquiring froma network phenotypic information associated with the subject. Theinformation can be stored in a database, e.g., a relational database. Inanother embodiment, the method further includes accessing the database,e.g., for records relating to other subjects, comparing the 52906,33408, or 12189 sequence of the subject to the 52906, 33408, or 12189sequences in the database to thereby determine whether the subject as a52906, 33408, or 12189-associated disease or disorder, or apre-disposition for such.

The present invention also provides in a network, a method fordetermining whether a subject has a 52906, 33408, or 12189 associateddisease or disorder or a pre-disposition to a 52906, 33408, or12189-associated disease or disorder associated with 52906, 33408, or12189, said method comprising the steps of receiving 52906, 33408, or12189 sequence information from the subject and/or information relatedthereto, receiving phenotypic information associated with the subject,acquiring information from the network corresponding to 52906, 33408, or12189 and/or corresponding to a 52906, 33408, or 12189-associateddisease or disorder (e.g., a disorder characterized by abnormal ion fluxsuch as a neurological disorder or a cardiac disorder), and based on oneor more of the phenotypic information, the 52906, 33408, or 12189information (e.g., sequence information and/or information relatedthereto), and the acquired information, determining whether the subjecthas a 52906, 33408, or 12189-associated disease or disorder or apre-disposition to a 52906, 33408, or 12189-associated disease ordisorder. The method may further comprise the step of recommending aparticular treatment for the disease, disorder or pre-disease condition.

The present invention also provides a method for determining whether asubject has a 52906, 33408, or 12189-associated disease or disorder or apre-disposition to a 52906, 33408, or 12189-associated disease ordisorder, said method comprising the steps of receiving informationrelated to 52906, 33408, or 12189 (e.g., sequence information and/orinformation related thereto), receiving phenotypic informationassociated with the subject, acquiring information from the networkrelated to 52906, 33408, or 12189 and/or related to a 52906, 33408, or12189-associated disease or disorder, and based on one or more of thephenotypic information, the 52906, 33408, or 12189. information, and theacquired information, determining whether the subject has a 52906,33408, or 12189-associated disease or disorder or a pre-disposition to a52906, 33408, or 12189-associated disease or disorder. The method mayfurther comprise the step of recommending a particular treatment for thedisease, disorder or pre-disease condition.

This invention is further illustrated by the following examples thatshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication are incorporated herein by reference.

EXAMPLES Example 1 Identification and Characterization of Human 52906,33408, and 12189 cDNAs

The human 52906 nucleic acid sequence is recited as follows:GCGTCCGCAGATTCCAGAGCCTGCCGGCTGGGAAAGA (SEQ ID NO:1)TCCGGTCTCGGGGTCGGCTATGATCCCGCAGCGGCCAAGGCAGGGCTCAGGCCCCGGGATTCTCCCCACACGCTGCTGCACTGGCGCAGCCGGTCGCCAAACTTTTTCTCCCCAAAGCCAGTGCCCCCGCAGTTACTTGGCGGGCAGCCGGCAGCCCACTCTCGGCGGGATGATCTGGGAGAAGCGGGCGTGGGACGAGGGGGCTGCTGTTTTGCAGCCCTGCGAGGCGTGCAGTCGGAGAAGTGGTCGGGGTTCCACACCGTCCCTGAGCCTGCCCCCTGGCCAAGGTGGCCCGACGTGCTGCAGTGGCTGGCGCAGGTGATCCGGGCAGCGCGTCCGGCACTAGTCAAGGGGGCAGCGGCACGGGAGGGAGGGGCGCCTTTCTCTTTTCTCCTCCCCCTGCAGCCCAGCTGCACTGCGTGGGGGCTCTCCATCTCCACGCAATCAGCAGGCGGAATCCCTGCCCTGGAGCGCCCTGGCTCTGGACTGCACCCCCCTAGGGTTTGTCCTGCAGATTCTCCTCCCCATCTTTCTCTGCCACACACGCTTCCCTAAGCCGCGCGCGCCGCAAACTCAGTCTCGGTCCCCGCAGGTGATGTCATGCCCATTGTTTTGGTGCGCCCAACCAATCGGACTCGCCGCCTGGATTCTACCGGAGCCGGCATGGGCCCTTCCTCGCACCAGCAGCAGGAGTCCCCGCTCCCGACCATAACGCATTGCGCAGGGTGCACCACCGCTTGGTCTCCCTGCAGCTTTAACAGCCCTGACATGGAAACCCCATTGCAGTTCCAGCGCGGCTTCTTCCCAGAGCAGCCGCCGCCGCCGCCGCGCTCCTCACACCTGCATTGCCAGCAGCAGCAACAGAGCCAGGACAAGCCGTGCCCGCCCTTCGCGCCCCTCCCGCACCCTCACCACCACCCGCACCTCGCGCACCAGCAGCCGGCCAGCGGCGGCAGCAGCCCATGCCTCCGGTGCAACAGCTGCGCCTCCTCCGGTGCCCCGGCGGCGGGGGCGGGAGATAACCTGTCCCTGCTGCTCCGCACCTCCTCGCCCGGCGGCGCCTTCCGGACCCGCACCTCCTCGCCGCTGTCGGGCTCGTCCTGCTGCTGCTGCTGCTGCTCGTCGCGCCGGGGCAGCCAGCTCAATGTGAGCGAGCTGACGCCGTCCAGCCATGCCAGTGCGCTCCGGCAGCAGTACGCGCAGCAGTCCGCGCAGCAGTCGGCGTCCGCCTCCCAGTACCACCAGTGCCACAGCCTGCAGCCCGCCGCCAGCCCCACGGGCAGCCTCGGCAGTCTGGGCTCCGGGCCCCCGCTCTCGCACCACCACCACCACCCGCACCCGGCGCACCACCAGCACCACCAGCCCCAGGCGCGCCGCGAGAGCAACCCCTTCACCGAAATAGCCATGAGCAGCTGCAGGTACAACGGGGGCGTCATGCGGCCGCTCAGCAACTTGAGCGCGTCCCGCCGGAACCTGCACGAGATGGACTCAGAGGCGCAGCCCCTGCAGCCCCCCGCGTCTGTCGGAGGAGGTGGCGGCGCGTCCTCCCCGTCTGCAGCCGCTGCCGCCGCCGCCGCTGTTTCGTCCTCAGCCCCCGAGATCGTGGTGTCTAAGCCCGAGCACAACAACTCCAACAACCTGGCGCTCTATGGAACCGGCGGCGGAGGCAGCACTGGAGGAGGCGGCGGCGGTGGCGGGAGCGGGCACGGCAGCAGCAGTGGCACCAAGTCCAGCAAAAAGAAAAACCAGAACATCGGCTACAAGCTGGGCCACCGGCGCGCCCTGTTCGAAAAGCGCAAGCGGCTCAGCGACTACGCGCTCATCTTCGGCATGTTCGGCATCGTGGTCATGGTCATCGAGACCGAGCTGTCGTGGGGCGCCTACGACAAGGCGTCGCTGTATTCCTTAGCTCTGAAATGCCTTATCAGTCTCTCCACGATCATCCTGCTCGGTCTGATCATCGTGTACCACGCCAGGGAAATACAGTTGTTCATGGTGGACAATGGAGCAGATGACTGGAGAATAGCCATGACTTATGAGCGTATTTTCTTCATCTGCTTGGAAATACTGGTGTGTGCTATTCATCCCATACCTGGGAATTATACATTCACATGGACGGCCCGGCTTGCCTTCTCCTATGCCCCATCCACAACCACCGCTGATGTGGATATTATTTTATCTATACCAATGTTCTTAAGACTCTATCTGATTGCCAGAGTCATGCTTTTACATAGCAAACTTTTCACTGATACCTCCTCTAGAAGCATTGGAGCACTTAATAAGATAAACTTCAATACACGTTTTGTTATGAAGACTTTAATGACTATATGCCCAGGAACTGTACTCTTGGTTTTTAGTATCTCATTATGGATAATTGCCGCATGGACTGTCCGAGCTTGTGAAAGGTACCATGATCAACAGGATGTTACTAGCAACTTCCTTGGAGCGATGTGGTTGATATCAATAACTTTTCTCTCCATTGGTTATGGTGACATGGTACCTAACACATACTGTGGAAAAGGAGTCTGCTTACTTACTGGAATTATGGGTGCTGGTTGCACAGCCCTGGTGGTAGCTGAGTGGCAAGGAAGCTAGAACTTACCAAAGCAGAAAAACACGTGCACAATTTCATGATGGATACTCAGCTGACTAAAAGAGTAAAAAATGCAGCTGCCAATGTACTCAGGGAAACATGGCTAATTTACAAAAATACAAAGCTAGTGAAAAAGATAGATCATGCAAAAGTAAGAAAACATCAACGAAAATTCCTGCAAGCTATTCATCAATTAAGAAGTGTAAAAATGGAGCAGAGGAAACTGAATGACCAAGCAAACACTTTGGTGGACTTGGCAAAGACCCAGAACATCATGTATGATATGATTTCTGACTTAAACGAAAGGAGTGAAGACTTCGAGAAGAGGATTGTTACCCTGGAAACAAAACTAGAGACTTTGATTGGTAGCATCCACGCCCTCCCTGGGCTCATAAGCCAGACCATCAGGCAGCAGCAGAGAGATTTCATTGAGGCTCAGATGGAGAGCTACGACAAGCACGTCACTTACAATGCTGAGCGGTCCCGGTCCTCGTCCAGGAGGCGGCGGTCCTCTTCCACAGCACCACCAACTTCATCAGAGAGTAGCTAGAAGAGAATAAGTTAACCACAAAATAAGACTTTTTGCCATCATATGGTCAATATTTTAGCTTTTATTGTAAAGCCCCTATGGTTCTAATCAGCGTTATCCGGGTTCTGATGTCAGAATCCTGGGAACCTGAACACTAAGTTTTAGGCCAAAATGAGTGAAAACTCTTTTTTTTTCTTTCAGATGCACAGGGAATGCACCTATTATTGCTATATAGATTGTTCCTCCTGTAATTTCACTAACTTTTTATTCATGCACTTCAAACAAACTTTACTACTACATTATATGATATATAATAAAAAAAGTTAATTTCTGCAAAAAAAAAAAAAAAAAAAAAA ACGGACGGG.

The human 52906sequence (FIG. 1; SEQ ID NO:1) is approximately 3525nucleotides long. The nucleic acid sequence includes an initiation codon(ATG) and a termination codon (TAG), which are underscored above. Theregion between and inclusive of the initiation codon and the terminationcodon is a methionine-initiated coding sequence of about 2544nucleotides, including the termination codon (nucleotides indicated as“coding” of SEQ ID NO:1; SEQ ID NO:3). The coding sequence encodes a 847amino acid protein (SEQ ID NO:2), which is recited as follows:MPIVLVRPTNRTRRLDSTGAGMGPSSHQQQESPLPTI (SEQ ID NO:2)THCAGCTTAWSPCSFNSPDMETPLQFQRGFFPEQPPPPPRSSHLHCQQQQQSQDKPCPPFAPLPHPHHHPHLAHQQPASGGSSPCLRCNSCASSGAPAAGAGDNLSLLLRTSSPGGAFRTRTSSPLSGSSCCCCCCSSRRGSQLNVSELTPSSHASALRQQYAQQSAQQSASASQYHQCHSLQPAASPTGSLGSLGSGPPLSHHHHHPHPAHHQHHQPQARRESNPFTEIAMSSCRYNGGVMRPLSNLSASRRNLHEMDSEAQPLQPPASVGGGGGASSPSAAAAAAAAVSSSAPEIVVSKPEHNNSNNLALYGTGGGGSTGGGGGGGGSGHGSSSGTKSSKKKNQNIGYKLGHRRALFEKRKRLSDYALIFGMFGIVVMVIETELSWGAYDKASLYSLALKCLISLSTIILLGLIIVYHAREIQLFMVDNGADDWRIAMTYERIFFICLEILVCAIHPIPGNYTFTWTARLAFSYAPSTTTADVDIILSIPMFLRLYLIARVMLLHSKLFTDTSSRSIGALNKINFNTRFVMKTLMTICPGTVLLVFSISLWIIAAWTVRACERYHDQQDVTSNFLGAMWLISITFLSIGYGDMVPNTYCGKGVCLLTGIMGAGCTALVVAVVARKLELTKAEKHVHNFMMDTQLTKRVKNAAANVLRETWLIYKNTKLVKKIDHAKVRKHQRKFLQAIHQLRSVKMEQRKLNDQANTLVDLAKTQNIMYDMISDLNERSEDFEKRIVTLETKLETLIGSIHALPGLISQTIRQQQRDFIEAQMES YDKHVTYNAERSRSSSRRRRSSSTAPPTSSESS.

The human 33408 nucleic acid sequence is recited as follows:GACCCACGCGTCCGCTCCCCCGTGTGCGGCACCGCCA (SEQ ID NO:4)CAGTCTGGGCAGCGGCGGCCGGGGGAGCGCTACTACCATGAACTGCCTGGTCCTCCTCCCCAGAGCTGCTCATCCGGGTCGGGCTGGAGACACAGTCAGGGGACCCCGTCGCCGCCGCCGCGCCCCCTCTTCTTTCGGCTCAATCTTCTCTTCCACCTTTTCCTCCTCTTCCTCCACCTTCTTTGCCTGCATCCCCCCCTCCCCCGCCGCGGATCCTGGCCGCTGCTCTCCAGACCCAGGATGCCGGGGGGCAAGAGAGGGCTGGTGGCACCGCAGAACACATTTTTGGAGAACATCGTCAGGCGCTCCAGTGAATCAAGTTTCTTACTGGGAAATGCCCAGATTGTGGATTGGCCTGTAGTTTATAGTAATGACGGTTTTTGTAAACTCTCTGGATATCATCGAGCTGACGTCATGCAGAAAAGCAGCACTTGCAGTTTTATGTATGGGGAATTGACTGACAAGAAGACCATTGAGAAAGTCAGGCAAACTTTTGACAACTACGAATCAAACTGCTTTGAAGTTCTTCTGTACAAGAAAAACAGAACCCCTGTTTGGTTTTATATGCAAATTGCACCAATAAGAAATGAACATGAAAAGGTGGTCTTGTTCCTGTGTACTTTCAAGGATATTACGTTGTTCAAACAGCCAATAGAGGATGATTCAACAAAAGGTTGGACGAAATTTGCCCGATTGACACGGGCTTTGACAAATAGCCGAAGTGTTTTGCAGCAGCTCACGCCAATGAATAAAACAGAGGTGGTCCATAAACATTCAAGACTAGCTGAAGTTCTTCAGCTGGGATCAGATATCCTTCCTCAGTATAAACAAGAAGCGCCAAAGACGCCACCACACATTATTTTACATTATTGTGCTTTTAAAACTACTTGGGATTGGGTGATTTTAATTCTTACCTTCTACACCGCCATTATGGTTCCTTATAATGTTTCCTTCAAAACAAAGCAGAACAACATAGCCTGGCTGGTACTGGATAGTGTGGTGGACGTTATTTTTCTGGTTGACATCGTTTTAAATTTTCACACGACTTTCGTGGGGCCCGGTGGAGAGGTCATTTCTGACCCTAAGCTCATAAGGATGAACTATCTGAAAACTTGGTTTGTGATCGATCTGCTGTCTTGTTTACCTTATGACATCATCAATGCCTTTGAAAATGTGGATGAGGGAATCAGCAGTCTCTTCAGTTCTTTAAAAGTGGTGCGTCTCTTACGACTGGGCCGTGTGGCTAGGAAACTGGACCATTACCTAGAATATGGAGCAGCAGTCCTCGTGCTCCTGGTGTGTGTGTTTGGACTGGTGGCCCACTGGCTGGCCTGCATATGGTATAGCATCGGAGACTACGAGGTCATTGATGAAGTCACTAACACCATCCAAATAGACAGTTGGCTCTACCAGCTGGCTTTGAGCATTGGGACTCCATATCGCTACAATACCAGTGCTGGGATATGGGAAGGAGGACCCAGCAAGGATTCATTGTACGTGTCCTCTCTCTACTTTACCATGACAAGCCTTACAACCATAGGATTTGGAAACATAGCTCCTACCACAGATGTGGAGAAGATGTTTTCGGTGGCTATGATGATGGTTGGCTCTCTTCTTTATGCAACTATTTTTGGAAATGTTACAACAATTTTCCAGCAAATGTATGCCAACACCAACCGATACCATGAGATGCTGAATAATGTACGGGACTTCCTAAAACTCTATCAGGTCCCAAAAGGCCTTAGTGAGCGAGTCATGGATTATATTGTCTCAACATGGTCCATGTCAAAAGGCATTGATACAGAAAAGGTCCTCTCCATCTGTCCCAAGGACATGAGAGCTGATATCTGTGTTCATCTAAACCGGAAGGTTTTTAATGAACATCCTGCTTTTCGATTGGCCAGCGATGGGTGTCTGCGCGCCTTGGCGGTAGAGTTCCAAACCATTCACTGTGCTCCCGGGGACCTCATTTACCATGCTGGAGAAAGTGTGGATGCCCTCTGCTTTGTGGTGTCAGGATCCTTGGAAGTCATCCAGGATGATGAGGTGGTGGCTATTTTAGGGAAGGGTGATGTATTTGGAGACATCTTCTGGAAGGAAACCACCCTTGCCCATGCATGTGCGAACGTCCGGGCACTGACGTACTGTGACCTACACATCATCAAGCGGGAAGCCTTGCTCAAAGTCCTGGACTTTTATACAGCTTTTGCAAACTCCTTCTCAAGGAATCTCACTCTTACTTGCAATCTGAGGAAACGGATCATCTTTCGTAAGATCAGTGATGTGAAGAAAGAGGAGGAGGAGCGCCTCCGGCAGAAGAATGAGGTGACCCTCAGCATTCCCGTGGACCACCCAGTCAGAAAGCTCTTCCAGAAGTTCAAGCAGCAGAAGGAGCTGCGGAATCAGGGCTCAACACAGGGTGACCCTGAGAGGAACCAACTCCAGGTAGAGAGCCGCTCCTTACAGAATGGAACCTCCATCACCGGAACCAGCGTGGTGACTGTGTCACAGATTACTCCCATTCAGACGTCTCTGGCCTATGTGAAAACCAGTGAATCCCTTAAGCAGAACAACCGTGATGCCATGGAACTCAAGCCCAACGGCGGTGCTGACCAAAAATGTCTCAAAGTCAACAGCCCAATAAGAATGAAGAATGGAAATGGAAAAGGGTGGCTGCGACTCAAGAATAATATGGGAGCCCATGAGGAGAAAAAGGAAGACTGGAATAATGTCACTAAAGCTGAGTCAATGGGGCTATTGTCTGAGGACCCCAAGAGCAGTGATTCAGAGAACAGTGTGACCAAAAACCCACTAAGGAAAACAGATTCTTGTGACAGTGGAATTACAAAAAGTGACCTTCGTTTGGATAAGGCTGGGGAGGCCCGAAGTCCGCTAGAGCACAGTCCCATCCAGGCTGATGCCAAGCACCCCTTTTATCCCATCCCCGAGCAGGCCTTACAGACCACACTGCAGGAAGTCAAACACGAACTCAAAGAGGACATCCAGCTGCTCAGCTGCAGAATGACTGCCCTAGAAAAGCAGGTGGCAGAAATTTTAAAAATACTGTCGGAAAAAAGCGTACCCCAGGCCTCATCTCCCAAATCCCAAATGCCACTCCAAGTACCCCCCCAGATACCATGTCAGGATATTTTTAGTGTCTCAAGGCCTGAATCACCTGAATCTGACAAAGATGAAATCCACTTTTAATATATATACATATATATTTGTTAATATATTAAAACAGTATATACATATGTGTGTATATACAGTATATACATATATATATTTTCACTTGCTTTCAAGATGATGACCACACATGGATTTTGATATGTAAATATTGCATGTCCAGCTGGATTCTGGCCTGCCAAAGAAGATGATGATTAAAAACATAGATATTGCTTGTATATTATGCAGTTGACTGCATGCACACTTTACATTTATTTATAATCTCTATTCTATAATAAAAGAGTATGATTTTTGTTAAAAAAAAAAAAAAAAAAAAAATTCCTCGCCGG A.

The human 33408 sequence (FIG. 3; SEQ ID NO:4) is approximately 3553nucleotides long. The nucleic acid sequence includes an initiation codon(ATG) and a termination codon (TAA), which are underscored above. Theregion between and inclusive of the initiation codon and the terminationcodon is a methionine-initiated coding sequence of about 2967nucleotides, including the termination codon (nucleotides indicated as“coding” of SEQ ID NO:4; SEQ ID NO:6). The coding sequence encodes a 988amino acid protein (SEQ ID NO:5), which is recited as follows:MPGGKRGLVAPQNTFLENIVRRSSESSFLLGNAQIVD (SEQ ID NO:5)WPVVYSNDGFCKLSGYHRADVMQKSSTCSFMYGELTDKKTIEKVRQTFDNYESNCFEVLLYKKRTPVWFYMQIAPIRNEHEKVVLFLCTFKDITLFKQPIEDDSTKGWTKFARLTRALTNSRSVLQQLTPMNKTEVVHKHSRLAEVLQLGSDILPQYKQEAPKTPPHIILHYCAFKTTWDWVILILTFYTAIMVPYNVSFKTKQNNIAWLVLDSVVDVIFLVDIVLNFHTTFVGPGGEVISDPKLIRMNYLKTWFVIDLLSCLPYDIINAFENVDEGISSLFSSLKVVRLLRLGRVARKLDHYLEYGAAVLVLLVCVFGLVAHWLACIWYSIGDYEVIDEVTNTIQIDSWLYQLALSIGTPYRYNTSAGIWEGGPSKDSLYVSSLYFTMTSLTTIGFGNIAPTTDVEKMFSVAMMMVGSLLYATIFGNVTTIFQQMYANTNRYHEMLNNVRDFLKLYQVPKGLSERVMDYIVSTWSMSKGIDTEKVLSICPKDMRADICVHLNRKVFNEHPAFRLASDGCLRALAVEFQTIHCAPGDLIYHAGESVDALCFVVSGSLEVIQDDEVVAILGKGDVFGDIFWKETTLAHACANVRALTYCDLHIIKREALLKVLDFYTAFANSFSRNLTLTCNLRKRIIFRKISDVKKEEEERLRQKNEVTLSIPVDHPVRKLFQKFKQQKELRNQGSTQGDPERNQLQVESRSLQNGTSITGTSVVTVSQITPIQTSLAYVKTSESLKQNNRDAMELKPNGGADQKCLKVNSPIRMKNGNGKGWLRLKNNMGAHEEKKEDWNNVTKAESMGLLSEDPKSSDSENSVTKNPLRKTDSCDSGITKSDLRLDKAGEARSPLEHSPIQADAKHPFYPIPEQALQTTLQEVKHELKEDIQLLSCRMTALEKQVAEILKILSEKSVPQASSPKSQMPLQVPP QIPCQDIFSVSRPESPESDKDEIHF.

The human 12189 nucleic acid sequence is recited as follows:TGCTGCGAGCGGCTGGTGCTCAACGTGGCCGGGCTGC (SEQ ID NO:7)GCTTCGAGACGCGGGCGCGCACGCTGGGCCGCTTCCCGGACACTCTGCTAGGGGACCCAGCGCGCCGCGGCCGCTTCTACGACGACGCGCGCCGCGAGTATTTCTTCGACCGGCACCGGCCCAGCTTCGACGCCGTGCTCTACTACTACCAGTCCGGTGGGCGGCTGCGGCGGCCGGCGCACGTGCCGCTCGACGTCTTCCTGGAAGAGGTGGCCTTCTACGGGCTGGGCGCGGCGGCCCTGGCACGCCTGCGCGAGGACGAGGGCTGCCCGGTGCCGCCCGAGCGCCCCCTGCCCCGCCGCGCCTTCGCCCGCCAGCTGTGCCTGCTTTTCGAGTTTCCCGAGAGCTCTCAGGCCGCGCGCGTGCTCGCCGTAGTCTCCGTGCTGGTCATCCTCGTCTCCATCGTCGTCTTCTGCCTCGAGACGCTGCCTGACTTCCGCGACGACCGCGACGGCACGGGGCTTGCTGCTGCAGCCGCAGCCGGCCCGTTCCCCGCTCCGCTGAATGGCTCCAGCCAAATGCCTGGAAATCCACCCCGCCTGCCCTTCAATGACCCGTTCTTCGTGGTGGAGACGCTGTGTATTTGTTGGTTCTCCTTTGAGCTGCTGGTACGCCTCCTGGTCTGTCCAAGCAAGGCTATCTTCTTCAAGAACGTGATGAACCTCATCGATTTTGTGGCTATCCTTCCCTACTTTGTGGCACTGGGCACCGAGCTGGCCCGGCAGCGAGGGGTGGGCCAGCAGGCCATGTCACTGGCCATCCTGAGAGTCATCCGATTGGTGCGTGTCTTCCGCATCTTCAAGCTGTCCCGGCACTCAAAGGGCCTGCAAATCTTGGGCCAGACGCTTCGGGCCTCCATGCGTGAGCTGGGCCTCCTCATCTTTTTCCTCTTCATCGGTGTGGTCCTCTTTTCCAGCGCCGTCTACTTTGCCGAAGTTGACCGGGTGGACTCCCATTTCACTAGCATCCCTGAGTCCTTCTGGTGGGCGGTAGTCACCATGACTACAGTTGGCTATGGAGACATGGCACCCGTCACTGTGGGTGGCAAGATAGTGGGCTCTCTGTGTGCCATTGCGGGCGTGCTGACTATTTCCCTGCCAGTGCCCGTCATTGTCTCCAATTTCAGCTACTTTTATCACCGGGAGACAGAGGGCGAAGAGGCTGGGATGTTCAGCCATGTGGACATGCAGCCTTGTGGCCCACTGGAGGGCAAGGCCAATGGGGGGCTGGTGGACGGGGAGGTACCTGAGCTACCACCTCCACTCTGGGCACCCCCAGGGAAACACCTGGTCACC GAAGTGTGA.

The human 12189 sequence (FIG. 5; SEQ ID NO:7) is approximately 1341nucleotides long. The nucleic acid sequence includes a termination codon(TGA), which is underscored above. The coding sequence encodes a 446amino acid protein (SEQ ID NO:8), which is recited as follows:CCERLVLNVAGLRFETRARTLGRFPDTLLGDPARRGR (SEQ ID NO:8)FYDDARREYFFDRHRPSFDAVLYYYQSGGRLRRPAHVPLDVFLEEVAFYGLGAAALARLREDEGCPVPPERPLPRRAFARQLCLLFEFPESSQAARVLAVVSVLVILVSIVVFCLETLPDFRDDRDGTGLAAAAAAGPFPAPLNGSSQMPGNPPRLPFNDPFFVVETLCICWFSFELLVRLLVCPSKAIFFKNVMNLIDFVAILPYFVALGTELARQRGVGQQAMSLAILRVIRLVRVFRIFKLSRHSKGLQILGQTLRASMRELGLLIFFLFIGVVLFSSAVYFAEVDRVDSHFTSIPESFWWAVVTMTTVGYGDMAPVTVGGKIVGSLCAIAGVLTISLPVPVIVSNFSYFYHRETEGEEAGMFSHVDMQPCGPLEGKANGGLVDGEVPELPPPLWAPPGKHLVT EV.

Example 2 Tissue Distribution of 52906 and 33408 mRNA by TaqMan Analysis

Endogenous human-52906 and 33408 gene expression was determined usingthe Perkin-Elmer/ABI 7700 Sequence Detection System which employs TaqMantechnology. Briefly, TaqMan technology relies on standard RT-PCR withthe addition of a third gene-specific oligonucleotide (referred to as aprobe) which has a fluorescent dye coupled to its 5′ end (typically6-FAM) and a quenching dye at the 3′ end (typically TAMRA). When thefluorescently tagged oligonucleotide is intact, the fluorescent signalfrom the 5′ dye is quenched. As PCR proceeds, the 5′ to 3′ nucleolyticactivity of Taq polymerase digests the labeled primer, producing a freenucleotide labeled with 6-FAM, which is now detected as a fluorescentsignal. The PCR cycle where fluorescence is first released and detectedis directly proportional to the starting amount of the gene of interestin the test sample, thus providing a quantitative measure of the initialtemplate concentration. Samples can be internally controlled by theaddition of a second set of primers/probe specific for a housekeepinggene such as GAPDH which has been labeled with a different fluorophoreon the 5′ end (typically VIC).

To determine the level of 52906 and 33408 in various human tissues aprimer/probe set was designed. Total RNA was prepared from a series ofhuman tissues using an RNeasy kit from Qiagen. First strand cDNA wasprepared from 1 μg total RNA using an oligo-dT primer and Superscript IIreverse transcriptase (Gibco/BRL). cDNA obtained from approximately 50ng total RNA was used per TaqMan reaction. Tissues tested include thehuman tissues and several cell lines shown in Tables 3 and 4. 52906 mRNAwas detected in brain, prostate tumor, and heart samples (Table 3).33408 expression was found in brain, heart, skin, and adipose samples(Table 4). TABLE 3 Expression of 52906 mRNA in Human Tissues and CellLines Tissue Relative Expression Artery/normal 0 Aorta/diseased 0Vein/normal 0 Coronary smooth muscle cells 0 Human umbilical veinendothelial cells 0 Hemangioma 0 Heart/normal 0 Heart/congestive heartfailure 0.1902 Kidney 0 Skeletal muscle 0 Adipose/normal 0 Pancreas 0Primary osteoblasts 0 Osteoclasts (differentiated) 0 Skin/normal 0Spinal cord/normal 0 Brain Cortex/normal 1.6367 BrainHypothalamus/normal 0 Nerve 0 Dorsal Root Ganglion 0 Breast/normal 0Breast/tumor 0 Ovary/normal 0 Ovary/tumor 0 Prostate/normal 0Prostate/tumor 1.1613 Salivary glands 0 Colon/normal 0 Colon/tumor 0Lung/normal 0 Lung/tumor 0 Lung/chronic obstructive pulmonary disease 0Colon/inflammatory bowel disease 0 Liver/normal 0 Liver fibrosis 0Spleen/normal 0 Tonsil/normal 0 Lymph node/normal 0 Smallintestine/normal 0 Macrophages 0 Synovium 0 Bone marrow/mononuclearcells 0 Activated peripheral blood mononuclear cells 0 Neutrophils 0Megakaryocytes 0 Erythroid cells 0 positive control 0

TABLE 4 Expression of 33408 mRNA in Human Tissues and Cell Lines TissueRelative Expression Prostate 0.00 Osteoclasts 0.00 Liver 0.00 Breast0.00 Breast 0.00 Skeletal Muscle 2.60 Skeletal Muscle 0.13 Brain 33.03Colon 0.06 Colon 0.01 Heart 30.71 Heart 0.00 Ovary 0.00 Ovary 0.00Kidney 0.00 Kidney 0.01 Lung 0.01 Lung 0.00 Vein 0.10 Vein 0.01 Adipose0.00 Adipose 4.26 Small Intestine 0.00 Thyroid 0.00 Bone Marrow 0.00Skin 11.72 Testes 0.37 Placenta 0.01 Fetal Liver 0.00 Fetal Liver 0.00Fetal Heart 0.00 Fetal Heart 0.00 Osteoblasts/undifferentiated 0.00Osteoblasts/differentiated 0.00 Osteoblasts/primary culture 0.00 SpinalCord 0.00 Cervix 0.00 Spleen 0.00 Spinal Cord 0.00 Thymus 0.00 Tonsil0.00 Lymph Node 0.00 Aorta 0.00

Example 3 Tissue Distribution of 52906, 33408, or 12189 mRNA by NorthernAnalysis

Northern blot hybridizations with various RNA samples can be performedunder standard conditions and washed under stringent conditions, i.e.,0.2×SSC at 65° C. A DNA probe corresponding to all or a portion of the52906, 33408, or 12189 cDNA (SEQ ID NO:1, SEQ ID NO:4, or SEQ ID NO:7)can be used. The DNA is radioactively labeled with ³²P-dCTP using thePrime-It Kit (Stratagene, La Jolla, Calif.) according to theinstructions of the supplier. Filters containing, for example, mRNA frommouse hematopoietic and endocrine tissues, and cancer cell lines(Clontech, Palo Alto, Calif.) can be probed in ExpressHyb hybridizationsolution (Clontech) and washed at high stringency according tomanufacturer's recommendations.

Example 4 Recombinant Expression of 52906, 33408, or 12189 in BacterialCells

In this example, 52906, 33408, or 12189 is expressed as a recombinantglutathione-S-transferase (GST) fusion polypeptide in E. coli and thefusion polypeptide is isolated and characterized. Specifically, 52906,33408, or 12189 is fused to GST and this fusion polypeptide is expressedin E. coli, e.g., strain PEB199. Expression of the GST-52906, 33408, or12189 fusion protein in PEB199 is induced with IPTG. The recombinantfusion polypeptide is purified from crude bacterial lysates of theinduced PEB199 strain by affinity chromatography on glutathione beads.Using polyacrylamide gel electrophoretic analysis of the polypeptidepurified from the bacterial lysates, the molecular weight of theresultant fusion polypeptide is determined.

Example 5 Expression of Recombinant 52906, 33408 or 12189 Protein in COSCells

To express the 52906, 33408, or 12189 gene in COS cells (e.g., COS-7cells, CV-1 origin SV40 cells; Gluzman (1981) CellI23:175-182), thepcDNA/Amp vector by Invitrogen Corporation (San Diego, Calif.) is used.This vector contains an SV40 origin of replication, an ampicillinresistance gene, an E. coli replication origin, a CMV promoter followedby a polylinker region, and an SV40 intron and polyadenylation site. ADNA fragment encoding the entire 52906, 33408, or 12189 protein and anHA tag (Wilson et al. (1984) Cell 37:767) or a FLAG tag fused in-frameto its 3′ end of the fragment is cloned into the polylinker region ofthe vector, thereby placing the expression of the recombinant proteinunder the control of the CMV promoter.

To construct the plasmid, the 52906, 33408, or 12189 DNA sequence isamplified by PCR using two primers. The 5′ primer contains therestriction site of interest followed by approximately twentynucleotides of the 52906, 33408, or 12189 coding sequence starting fromthe initiation codon; the 3′ end sequence contains complementarysequences to the other restriction site of interest, a translation stopcodon, the HA tag or FLAG tag and the last 20 nucleotides of the 52906,33408, or 12189 coding sequence. The PCR amplified fragment and thepCDNA/Amp vector are digested with the appropriate restriction enzymesand the vector is dephosphorylated using the CIAP enzyme (New EnglandBiolabs, Beverly, Mass.). Preferably the two restriction sites chosenare different so that the 52906, 33408, or 12189 gene is inserted in thecorrect orientation. The ligation mixture is transformed into E. colicells (strains HB101, DH5α, SURE, available from Stratagene CloningSystems, La Jolla, Calif., can be used), the transformed culture isplated on ampicillin media plates, and resistant colonies are selected.Plasmid DNA is isolated from transformants and examined by restrictionanalysis for the presence of the correct fragment.

COS cells are subsequently transfected with the 52906, 33408, or12189-pcDNA/Amp plasmid DNA using the calcium phosphate or calciumchloride co-precipitation methods, DEAE-dextran-mediated transfection,lipofection, or electroporation. Other suitable methods for transfectinghost cells can be found in Sambrook, J., Fritsh, E. F., and Maniatis, T.(1989) Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold SpringHarbor Laboratory, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. The expression of the 52906, 33408, or 12189 polypeptide isdetected by radiolabelling (³⁵S-methionine or ³⁵S-cysteine availablefrom NEN, Boston, Mass., can be used) and immunoprecipitation (Harlow,E. and Lane, D. (1988) Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y.) using an HA specificmonoclonal antibody. Briefly, the cells are labeled for 8 hours with³⁵S-methionine (or ³⁵S-cysteine). The culture media are then collectedand the cells are lysed using detergents (RIPA buffer, 150 mM NaCl, 1%NP40, 0.1% SDS, 0.5% DOC, 50 mM Tris, pH 7.5). Both the cell lysate andthe culture media are precipitated with an HA specific monoclonalantibody. Precipitated polypeptides are then analyzed by SDS-PAGE.

Alternatively, DNA containing the 52906, 33408, or 12189 coding sequenceis cloned directly into the polylinker of the pCDNA/Amp vector using theappropriate restriction sites. The resulting plasmid is transfected intoCOS cells in the manner described above, and the expression of the52906, 33408, or 12189 polypeptide is detected by radiolabelling andimmunoprecipitation using a 52906, 33408, or 12189 specific monoclonalantibody.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1-7. (Canceled)
 8. An isolated polypeptide selected from the groupconsisting of: a) a polypeptide which is encoded by a nucleic acidmolecule comprising a nucleotide sequence which is at least 95%identical to a nucleic acid comprising the nucleotide sequence of SEQ IDNO:1 or SEQ ID NO:3 or at least 90% identical to a nucleic acidcomprising the nucleotide sequence of SEQ ID NO:7; b) a naturallyoccurring allelic variant of a polypeptide comprising the amino acidsequence of SEQ ID NO:2 or SEQ ID NO:8, wherein the polypeptide isencoded by a nucleic acid molecule which hybridizes to a nucleic acidmolecule comprising SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:7, or acomplement thereof under stringent conditions; and c) a fragment of apolypeptide comprising the amino acid sequence of SEQ ID NO:2 or SEQ IDNO:8, wherein the fragment comprises at least 580 contiguous amino acidsof SEQ ID NO:2 or at least 80 contiguous amino acids of SEQ ID NO:8. 9.The isolated polypeptide of claim 8, comprising the amino acid sequenceof SEQ ID NO:2 or SEQ ID NO:8.
 10. The polypeptide of claim 8, furthercomprising a heterologous amino acid sequence.
 11. An antibody whichselectively binds to the polypeptide of claim
 8. 12. (Canceled)
 13. Amethod for detecting the presence of a polypeptide of claim 8 in asample, comprising: a) contacting the sample with a compound whichselectively binds to a polypeptide of claim 8; and b) determiningwhether the compound binds to the polypeptide in the sample.
 14. Themethod of claim 13, wherein the compound which binds to the polypeptideis an antibody.
 15. A kit comprising a compound which selectively bindsto a polypeptide of claim 8 and instructions for use. 16-18. (Canceled)19. A method for identifying a compound which binds to a polypeptide ofclaim 8 comprising the steps of: a) contacting a polypeptide, or a cellexpressing a polypeptide of claim 8 with a test compound; and b)determining whether the polypeptide binds to the test compound.
 20. Themethod of claim 19, wherein the binding of the test compound to thepolypeptide is detected by a method selected from the group consistingof: a) detection of binding by direct detecting of testcompound/polypeptide binding; b) detection of binding using acompetition binding assay; c) detection of binding using an assay for52906 or 12189-mediated signal transduction.
 21. A method for modulatingthe activity of a polypeptide of claim 8 comprising contacting apolypeptide or a cell expressing a polypeptide of claim 8 with acompound which binds to the polypeptide in a sufficient concentration tomodulate the activity of the polypeptide.
 22. A method for identifying acompound which modulates the activity of a polypeptide of claim 8,comprising: a) contacting a polypeptide of claim 8 with a test compound;and b) determining the effect of the test compound on the activity ofthe polypeptide to thereby identify a compound which modulates theactivity of the polypeptide.
 23. A method of treating or preventing anion flux-related disorder in a subject, the method comprisingadministering to the subject an agent that modulates the activity orexpression of a 52906 or 12189 polypeptide or nucleic acid, in an amounteffective to treat or prevent the ion flux-related disorder.
 24. Themethod of claim 23, wherein the agent is a peptide, a phosphopeptide, asmall molecule, an antibody, or any combination thereof.
 25. The methodof claim 23, wherein the agent is an antisense, a ribozyme, a triplehelix molecule, a 52906 or 12189 nucleic acid, or any combinationthereof.
 26. A method for identifying an agent that modulates theactivity or expression of a 52906 or 12189 polypeptide or nucleic acid,comprising contacting the 52906 or 12189 polypeptide or nucleic acidwith an agent, and determining the effect of the agent on the activityor expression of the polypeptide or nucleic acid.
 27. The method ofclaim 26, wherein the agent is a peptide, a phosphopeptide, a smallmolecule, an antibody, or any combination thereof.
 28. The method ofclaim 26, wherein the agent is an antisense, a ribozyme, a triple helixmolecule, a 52906 or 12189 nucleic acid, or any combination thereof. 29.The method of claim 26, wherein the method comprises determining theeffect of the agent on an ion channel activity of the polypeptide. 30.The method of claim 26, wherein the effect of the agent on the activityor expression of the polypeptide or nucleic acid is determined in aneuronal cell or a muscle cell.
 31. An antibody which selectively bindsto an extracellular domain of the polypeptide of SEQ ID NO:5.